MXPA01006483A - Methods and compositions for treatment of cell proliferative disorders - Google Patents

Abstract

The present invention relates to methods and compositions comprising at least one endothelin antagonist, preferably in combination with a poly-b-1®-4-N-acetylglucosamine (p-G1cNAc) polysaccharide matrix, for use in the treatment of cancer and other proliferative diseases. The endothelin antagonist can be a peptide or non-peptide compound, and the p-G1cNAc matrix of the invention is comprised of a polymer of high molecular weight whose constituent monosaccharide sugars are attached in a b-1®-4 conformation, and which is free of proteins, and substantially free of single amino acids, and other organic and inorganic contaminants. The compositions and methods of the invention are useful for inhibiting the growth of tumors and other neoplastic cells and/or for inhibiting the metastasis of neoplastic cells i(in vivo).

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF DISORDERS OF CELL PROLIFERATION The present application is a continuation in part of the US application Serial No. 09 / 218,288, filed on December 22, 1988, which is a continuation in part of the application US Serial No. 471,290, filed June 6, 1995, now US Patent No. 5,858,350, said application is a continuation in part of US Patent Application Serial No. 347,911 filed on December 1, 1994 now U.S. Patent No. 5,623,064, which is a continuation in part of the application of US Patent No. 160,569, filed December 1, 1993, now US Patent No. 5,622,834, said applications are hereby incorporated by reference in their entireties . 1. INTRODUCTION The present invention relates to methods and compositions comprising at least one endothelin antagonist, preferably in combination with a polysaccharide matrix of poly-β-l- > 4-N-acetylglucosamine (p-GlcNAc), for use in the treatment of cancer and other cell proliferation diseases. More specifically, the endothelin antagonist of the invention can be a peptide or a non-peptide compound, and the p-GlcNAc matrix of the present invention consists of a high molecular weight polymer whose constituent monosaccharide sugars are fixed in the β-1-4 conformation, and without proteins, and substantially free of individual amino acids or other organic and inorganic contaminants. The compositions and methods of the invention are useful for inhibiting the growth of tumors and other neoplastic cells and / or for inhibiting the metastasis of neoplastic cells in vivo. 2. BACKGROUND OF THE INVENTION Endotelins are a family of peptides of 21 amino acids, for example, ET-1, ET-2, and ET-3, originally characterized by their potent vasoconstrictive and angiogenic properties (see for example, Luscher et al. ., 1995, Agents Actions Suppl. (Switzerland) 45: 237-253; Yanagisa et al., 1988 Nature 332: 411-415). These peptides appear additionally to be related to growth factors such as bFGF and often act in synergy with them (see, for example, Halaban, 1996, Seminars in Oncology 23: 673-681; Reid et al., 1996, Development 122: 3911 -3919; Markewitz et al., 1995, Am. J. Physiol. 268: L192-L200; and Nelson et al., 1996, Cancer Res. 56: 663-668). In addition, these peptides have regulatory properties of the cytokine type and can be influenced by hormones such as insulin and angiotensin II as well as by growth factors such as TGF-β and TNF-α (Nelson et al., Supra; Suzuki et al., 1989, J. Biochem. 106: 736-741; and Lundblad et al., 1996, Crit. Care Med. 24: 820-826). The activity of endothelin is mediated through the linkage with preferential affinities by two different G-coupled receptors, ETA and ETB, in autocrine / paracrine form (see, for example, Hocher et al., 1997, Eur. J. Clin. Chem. Clin Biochem.35 (3): 175-189; Shichiri et al., 1991, J. Cardiovascular Pharmacol. 17: S76-S78). There are several agonists and antagonists of endothelin receptors (Webb et al., 1997, Medicinal Research Reviews 17 (l): 17-67), which have been used to study the mechanism of action of endothelins. Since endothelin is known to have potent vasoconstrictor activity, endothelin antagonists, in particular, (also known as "endothelin receptor antagonists" in the art) have been studied for their possible role in the treatment of diseases. human, more specifically cardiovascular diseases such as hypertension, congestive heart failure, atherosclerosis, restenosis and myocardial infarction (Mateo et al., 1997, Pharmacological Res. 36 (5): 339-351). For example, endothelin antagonists not based on peptides belonging to the pyrimidinyl sulfonamide family, such as Ro 46-2005 and bosentan, which interact with the endothelin receptor through its aromatic rings, are currently in the process of clinical evaluation for the treatment of hypertension, vascular disease, as well as congestive heart failure. These antagonists can bind both ETA and ETB with varying affinities and have advantages compared to peptide-based antagonists since they possess improved metabolic stability (Webb et al., Supra; and Parris et al., Supra). In addition, endothelin antagonists have also been studied in terms of their possible role in the treatment of kidney disease such as impaired renal function in liver cirrhosis and acute renal failure (Gómez-Garre et al., 1996, Kicney Int. 50: 962-972); Hocher et al., Supra). More recently, endothelin and endothelin receptors have been implicated in numerous processes of normal and pathological cell growth, for example, cell cycle progression, cell growth, cell development (see, for example, Parris et al., 1997, Vascular Medicine 2: 31-43; Markewitz et al., Supra; Morbidelli et al., 1995, Am. J. Physiol. 269: H686-H695; and Battistini et al. 1993, Peptides 14: 385-399). It has been shown that ET1 and ET3 are mitogenic and chemokinetic factors for normal tissues that are within a range of endothelial and epithelial cells to macrophages (see, for example, Webb et al., 1997, Medicinal Research Reviews 17 (l): 17-67; and Gomez-Garre et al., Supra). In addition, the endothelin binding with its receptors causes DNA synthesis, proliferation and cell mobilization in normal and neoplastic cells (Webb et al., Supra; Ziche et al., 1995, Cardiovasc.

Pharmacol. 26: S284-S286; and Yamashita et al., 1991, Res. Comm. in Chem. Pathol. and Pharmacol. 74 (3): 363-369). This potential ability of endothelins to mediate cell growth and cell cycle progression has led to some initial studies of endothelin expansion and / or endothelin receptor presence in cancer cells. For example, it has been shown that ET-1 is overexpressed in breast cancer and pancreatic cell lines and induces proliferation in breast cancer tissue, ovarian cell lines and prostate tumors, (see, for example, Moriatis et al., 1997, Eur. J. Canc 33 (4): 661-668; Nelson et al., 1996, Cancer Res. 56: 663-668; Patel et al., 1995 , Br. J. Cancer 71: 442-447; Oikawa et al., 1994, Br. J. Cancer 69: 1059-1064; Shichiri et al., Supra; and Yamashita et al., Supra). In addition, the presence of ETA-type receptors that have a higher affinity for ET1 and ET2, has been demonstrated in ovarian cell lines (Moriatis et al., Supra) and breast cancer tissues (Yamashita et al., Supra) . One of the few tumors that express ETB receptors that have a similar affinity for the three isoforms of endothelin is melanoma (Yohn et al., 1994, Biochem, Biochem, Biophys, Res. Comm. 201 (1): 449-457). ). By way of Interestingly, ETB receptors are expressed in important ways in primary or recurrent melanomas but less in metastatic melanomas (Kikuchi et al., 1996, Biochem, Biophys, Res.Comm. 219: 734-739). Even though these studies suggest that endothelin antagonists could potentially have therapeutic applications in the treatment of cancer, there have been no studies to date that demonstrate such therapeutic application. In fact, the role of endothelin in the promotion of a proliferative disease such as several vascular proliferative diseases and benign prostatic hypertrophy (BPH) is not clear (Webb et al., Supra and Kenny et al., 1997, J. Med. Chem. 40 (9): 1293-1315). In addition, while U.S. Patent Nos. 5,550,110 and 5,641,752 disclose the use of specific hexapeptide endothelin antagonists for the treatment of cancer, in fact there is no data in these disclosures as to: o the treatment of cancer and there are no indications as to how to effect said treatment or whether said treatment could be successful (see also, PCT applications WO 97/37987, 97/08169, WO 96/11927, and WO 94/03483, Canadian Patent Application No. 2072395, and US Patent No 5, 658, 943). 3. COMPENDIUM OF THE INVENTION The present invention relates to methods and compositions for the treatment of cell proliferation disorders such as cancer. More specifically, the invention relates to compositions comprising at least one endothelin antagonist, preferably in combination with a poly-β-l- 4-N-acetylglucosamine (p-GlcNAc) polyaccharide matrix, for Use in the treatment of cancer and other proliferative diseases. The present invention is based, in part, upon the discovery of the Applicants in that, when an endothelin antagonist is administered in vivo, either only in high doses or in combination with a polysarbaryl matrix, it is inhibited. growth of tumor cells and / or the growth or metastasis of neoplastic cells significantly. According to a preferred embodiment of the invention, the endothelin antagonist is a non-peptide-based pyriridyl sulfonamide compound, such as that shown in Figure I below.

Na "N ^ NH N = N The compound of Figure I is the sodium salt of [6- (2-hydroxy-ethoxy) -5- (2-methoxy-phenoxy), -2- [2 (1H-tetrazol-5-yl) -pyridin-4 5-isopropyl-pyridine-2-sulfonic acid (1: 2), which is also referred to herein as "Rodl" and has a molecular weight of approximately 650 kD. It is a non-specific inhibitor, not a peptide of both endothelin receptors, ETA and ETB. In accordance with a preferred embodiment of the invention, the matri: The polyaccharide is a poly-β-1-4-N-acetylglucosamine (p-GlcNAc) polyacid matrix, or a derivative thereof, in accordance with that described in US Patent No. 5,635,493, which is incorporated herein by reference. here by reference in its entirety. P-GlcNAc or its derivatives can be used in several reformulations, including membranes, filaments, nonwovens, sponges, gels as well as three-dimensional matrices. * In accordance with a preferred embodiment, the p-GlcNAc is in the form of a gel, is preferably deacetylated and optionally derived to a lactate salt of p-GlcNAc and is combined with Ro61 for in vivo administration. The compositions of the invention are useful for drug delivery systems, e.g., slow release drug administration. The compositions of the invention are an improvement compared to traditional pharmacological formulations insofar as the Compositions of the present invention offer, for example, greater effectiveness, lower toxicity, and greater bioavailability. The methods of the invention comprise the administration of therapeutically effective amounts of the compositions of the invention in vivo for the treatment of cell proliferation diseases, for example, cancer in an animal, including humans. According to one embodiment of the invention, for example an endothelin antagonist, such as for example R06I, deacetylated p-GlcNAc lactate gel is dissolved and administered in a therapeutically effective amount to a patient in vivo for the treatment of cancer or well other proliferative diseases or proliferative disorders. Another embodiment of the invention comprises the in vivo administration of an endothelin antagonist, more preferably, a non-peptide-based endothelin antagonist, such as an endothelin antagonist of pyrimidyl sulfonairide, for the treatment of cancer or other diseases or disorders of proliferation. Another embodiment of the present invention encompasses the in vivo administration of a p-GlcNAc matrix alone for the treatment of cancer or other proliferative diseases or disorders. The compositions and methods of the invention are useful for the inhibition of tumor and / or other neoplastic cell growth. and / or for the inhibition of neoplastic cell metastasis in vivo. 4. BRIEF DESCRIPTION OF THE FIGURES FIGURE 1. Chemical structure of 100% p-GlcNAc. "n" refers to an integer within a range of approximately 4,000 to about 150,000, with between about 4,000 and about 15,000 being preferred. FIGURE 2. Analysis of carbohydrate of p-GlcNAc, mass spectroscopy data-gas chromatography. The filled squares represent purified p-GlcNAc by using the acid / neutralization treatment method described in section 5.1, infra. FIGURE 3. Scanning electron micrograph representing a p-GlcNAc membrane prepared by variation of acid treatment / neutralization of the chemical / biological purification method. Amplification: 10,000 times.

FIGURE .: . Diagram showing some of the possible p-GlcNAc and possible deacetylated derivatives of p-GlcNAc of the invention (adapted from S. Hirano, in "Chitin and Chitosan", 1989, Skjak-Braek, Anthonsen, and Sanford, eds., Elsevier Science Publishing Co., pages 37-43). FIGURES 5A and 5B. Scanning electron micrographs of a deacetylated p-GlcNAc mat. Amplification: FIGURE 5A: 1000 times; FIGURE 5B: 10,000 times. FIGURES 6A and 6B. Scanning electron micrographs of a p-GlcNAc membrane dissolved in dimethylacetamide / lithium chloride and again mentioned in water in a fibrous material, according to that described in the section of Example 8, infra. FIGURE 7. Inhibition of endothelin receptor antagonist Rodl of proliferation of B16 melanoma cells in vitro. Ro61 was added in increasing concentrations to a 96-well culture plate to which B16 cells (filled circles) and splenocytes (open circles) were added from C57BL / 6 (H-2b) mice. The proliferation of cells treated with Ro61 is expressed as a percentage of untreated control cells. The mean values of the wells were determined in triplicate. FIGURE 8. Bar graphs indicating the percentage proliferation of B16 melanoma cells compared to untreated controls when the cells were exposed to several endothelin antagonists. The results indicate an inhibition of proliferation upon exposure of the cells to endothelin antagonists. FO cells are control B16 cells that do not have a full-length ETA receptor; the bar graph labeled ET1 represents a control, where the cells were exposed to a known agonist of endothelin. FIGURE 9. Bar graphs indicating the percentage of proliferation of B16 melanoma cells compared to untreated controls when the cells are exposed to several endothelin antagonists. The results indicate an inhibition of proliferation upon exposure to endothelin antagonists. FO cells are control B16 cells that do not have a full length ETA receptor; the bar graphs labeled ET1 and BQ3020 represent controls, where the cells were exposed to two known agonists of endothelin. FIGURE 10. Reversal with ETA and ETB agonists of the inhibition by Rodl of the proliferation of B16 melanoma cells in vitro. Celulsa B16 were cultured with either agonists BQ-3020- [Ac- [Alall, Alal5] -endothelin (6, 21) (filled triangles), agonists [Ala "'"' 1"" 0] -endotelinal (empty rhombus), both agonists (empty square) or none (full circle) and Ro61 was then added to each well. The proliferation of the cells treated with Rodl is expressed as the percentage of untreated control cells. The mean values of the wells were determined in triplicate. FIGURES HA and 11B. Effect of Ro61 on B16 cells in culture. Light micrograph of B16 cells with a 40-fold amplification. FIGURE A: B16 cells cultured in 96-well plates at 5x144 cells / well for 72 hours at a temperature of 37 ° C in complete medium; FIGURE B: B16 cells cultured in complete medium containing 5 μM Ro61.

FIGURE 12. R06I induces apoptosis. B16 cells were assayed for apoptosis with a Fluorescein In Situ Cell Death Detection Kit after culture either with common medium (control) or with R06I (1 uM) (I) for 0, 24, 48 and 72 hours at a temperature of 37 ° C. FIGURES 13A and 13B. Inhibition by R06I of intraperitoneal carcinomatosis B16. FIGURE 13A shows the effect of IP R06I on intraperitoneal B16 carcinomatosis. Female C57BL / 6 mice received IP injections with 5 x 10"" B16 melanoma cells. The following day the animals received injections of 100 μl of either: daily x 6 HBSS (control), daily x 6 HBSS containing 3 mg / kg of R06I (low dose), daily x 6 HBSS containing 30 mg / kg of R06I (high dose). FIGURE 13B shows the effect of administering p-GlcNAc from Rodl. The animals were challenged with tumor as in FIGURE 13A and treated the next day with 100 μl of p-GlcNAc gel injected either IP, SC or IP with 18 mg / kg of R06I (IP + R06I). The animals were sacrificed after 7 days and evaluated for the presence of B16 colonies. The values represent the average number of visible colonies and the standard error for each group (n = 11 for all groups, except group without treatment, n = 13).

FIGURE 14. Delayed appearance of tumors in C57BL / 6 mice treated with Rodl after a challenge with subcutaneous B16 melanoma type tumor. Female C57BL / 6 mice received an SC injection of 5 x 104 B16 melanoma cells. The following day the animals were randomly separated into four groups: no treatment (A), an IP injection of p-GlcNAc gel alone (0), daily IP injections of 3 mg / kg (for 6 days), of Rodl in HBSS) an IP injection of p-GlcNAc gel containing 18 mg / kg of Rodl (I). The animals were monitored for the presence of tumor for 3 weeks (n = 10 in all groups). FIGURE 15. Inhibitory effect of several endothelin antagonists in relation to the appearance of B16 tumor colonies using the carcinomatosis model described infra. FIO represents untreated B16 control cells; Bqmix represents a mixture of the ETA antagonist, BQ123 and ETB antagonists, BQ788; Bqmix / gel represents a mixture of BQ123 and BQ788 in combination with the p-GlcNAc gel described infra; Rodl is the non-specific ETA / ETB endothelin antagonist described infra; Ro61 / fel is Rool in combination with p-GlcNAc; and GRGDS / gel is a combination of the endothelin antagonist ETA / ETB GRGDS in combination with p-GlcNAc. FIGURE 16. Long-term survival of C57BL / 6 mice treated with Rodl after a intraperitoneal challenge with melanoma B16. C57BL / 6 mice received intraperitoneal injections with B16 cells. The animals were randomly separated into four groups for any of the following treatments: (a) untreated (squares filled); (b) 100 μl of p-GlcNAc gel alone (crosses); (c) 100 μl of HBSS daily containing 3 mg / kg of Rodl (filled triangles); or (d) 100 μl of p-GlcNAc gel containing 18 mg / kg of Rodl (empty squares). The animals were monitored daily and slaughtered for humanitarian reasons when it was determined that they were dying. FIGURES 17A and 17B. Microphotographs of the cell morphology of B16 cells treated with Rodl (FIGURE 17A) and untreated (FIGURE 17B) at a 40-fold magnification; 10"7M Ro61, 105 cells 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to compositions comprising at least one endothelin antagonist, preferably in combination with a poly-β-1-polyaccharide matrix. 4-N-acetylglucosamine (p-GlcNAc), and methods for using these compositions in the treatment of cancer and other proliferative diseases.The endothelin antagonists according to this invention can be specific or non-specific for ETA or ETB receptors. or compounds based on peptides or not based on peptides. According to a preferred embodiment of the invention, the endothelin antagonist is a non-specific, non-peptide-based endothelin antagonist. According to another preferred embodiment, the endothelin antagonist is a pyrimidyl sulfonamide compound not based on peptide, such as for example the compound Rodl illustrated in Figure I below.

In accordance with the present invention, at least one type of endothelin antagonist, alone or in combination with one or more other antitumor agents, is fixed covalently or non-covalently with the p-GlcNac described in detail in Section 5.1, infra or is combined with said p-GlcNAc. According to a preferred embodiment of the invention, at least one endothelin antagonist, such as Ro61, is dissolved in a p-GlcNAc gel. deacetylated to form an endothelin ("EA") / p-GlcNAc antagonist composition of the invention. According to a further preferred embodiment, the deacetylated p-GlcNAc is derived with lactic acid to form a lactate salt of p-GlcNAC. As defined herein, the term "endo'celin antagonist" includes endothelin receptor antagonists and "EA / p-GlcNAc compositions" include compositions wherein at least one type of endothelin antagonist is either covalently fixed. on p-GlcNAc is either non-covalently bound or mixed or encapsulated with p-GlcNAc. The compositions of the present invention may additionally comprise other antitumor agents, which, in combination with the endothelial antagonist, act to inhibit tumor growth and / or metastasis or other neoplastic cells. As defined herein, the "antitumor agent" includes any compound that inhibits the growth or metastasis of tumor cells, cancer cells, or any other type of neoplastic cell. This invention is based, in part, on the discovery of the Applicants, in the sense that endothelin antagonists, alone or in combination with the p-GlcNAc described herein, inhibit the proliferation of neoplastic cells in vitro and decrease metastasis and / or increase the survival of animals carrying tumor cells in vivo (see Example Sections 12 to 16, infra). In addition, the p-GlcNAc of this invention alone has an inhibitory effect on the metastasis and growth of neoplastic cells in vivo. Thus, according to the methods of the invention, pharmaceutical compositions comprising the compositions EA / p-GlcNAc in a therapeutically effective amount, are administered to a patient in vivo for the treatment of cancer or other proliferative diseases. Another preferred embodiment of the invention comprises the in vivo administration of an endothelin antagonist, for example, an endothelin pyrimidyl sulfanamide antagonist for the treatment of a proliferative disease. And, another embodiment includes the in vivo administration of the p-GlcNAc described infra for the treatment of a proliferative disease. Just to facilitate the description, the detailed description of this invention is divided into the following subsections: (1) the p-GlcNAc of the compositions and methods of the invention; (2) endothelin antagonists of the compositions and methods of the invention; (3) preferred formulations of the compositions of the invention; and (4) uses of the compositions and methods of the invention. 5.1 The p-GlcNAc of the compositions of the invention The polyaccharide matrix of p-GlcNAc to be used in the compositions and methods of this invention comprises a high molecular weight polymer which ranges from about 800,000 daltons to about 30 million daltons, based on chromatography measurements of gel permeation. Said molecular weight range represents a species of p-GlcNAc having from about 4,000 to about 150,000 N-acetylglucosamine monosaccharides fixed in a β-1-4 configuration, preferring from about 4,000 to about 15,000 N-acetylglucosamine monosaccharides (Figure 1). The variability of p-GlcNAc is very low, and its purity is very high, which is evidenced by chemical and physical criteria. Among these criteria are the chemical composition and the contaminants that are not polisácaridos. First, the chemical composition data for the p-GlcNAc produced using two different purification methods are shown in Table I. As can be seen, the chemical composition of the p-GlcNAc produced by both methods is, within the limits of experimental error, equal to the formula compositions of p-GlcNAc. Second, as 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 of the present invention can deviate to about 2% of the theoretical values of carbon, hydrogen, nitrogen and oxygen for a pure p-GlcNAc). Therefore, as used here, the terms "substantially free of organic contaminants" and "substantially free of inorganic contaminants" refer to p-GlcNAc compositions having the carbon, hydrogen, nitrogen and oxygen profiles that deviate by no more than about 2% of the theoretical values, and preferably, p-GlcNAc of the invention contains a conformance profile with that exemplified in the experimental data on p-GlcNAc mats in Table I (allowing percentage deviation). In addition, p-GlcNAc shows a very low percentage of bound water. TABLE I Chemical Analysis Data (% in pesos) Theoretical values of pure pGlcNAc Carbon - 47.29 Hydrogen - 6.40 Nitrogen - 6.89 Oxygen - 39.41 Protein - 0.00 Ein experimental data in p-GlcNAc mats: (the number of experimental batches for each type of membrane is greater than 30 for each type of membrane) Mechanical strength method Chemical / biological method Normalized% Dev. Normalized% Dev. Coal 47.21 + 0.08 - 0.17 47.31 + 0.11 + 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 Values Average Values Average Proteins 0.00 0 ., 00 Ash 1.30 0..98 Humidity 2.0 1. .2 1 The raw analytical data have been standardized to take into account the ash and moie content of the samples. The p-GlcNAc of the compositions of the invention has a carbohydrate analysis profile substantially similar to the profile presented in Figure 2. The primary monosaccharide of p-GlcNAc is N-acetylglucosamine. In addition, p-GlcNAc does not contain the monosaccharide glucosamine. Other physical characteristics of p-GlcNAc are described in detail in U.S. Patent No. 5,635,493, which has been incorporated herein by reference. The p-GlcNAc according to this invention presents a high degree of biocompatibility, which can be determined through various techniques, including, but not limited to, procedures such as elusion test, intramuscular implant, or intracutaneous or systemic injection in animal subjects. See, for example, U.S. Patent No. 5,635,493 which is incorporated herein by reference. P-GlcNAc is produced from microalgae and can be purified from said microalgae, preferably diatoms. Diatoms that can be used as initial sources for the production of p-GlcNAc include, but are not limited to, members of the genus Coscinodiscus, of the genus Cyclotella, and of the genus Thalassiosira, with the Thalassiosira genus being preferred. Among the genus Coscinodiscus, the diatom species that can be used include, but are not limited to, the concinnus and radiatus species. Diatoms within the Cyclotella genus that may be employed include, but are not limited to, the Caspian, Cryptic and Meneghinian species. The diatoms of the genus Thalassiosira which can be used to produce the initial material for the p-GlcNAc of this invention include, but are not limited to, the species nitzschoides, aestivalis, antarctica, deciphens, eccentnca, floridana, fluviatilis, gravid, guillardii, hyalina, minimum, nordenskioldii, oceanic, polychorda, pseudonana; rotula, tubifera, túmida, and weissflogii, preferring the species fluviatilis and weissflogii. Diatoms such as those described above can be obtained, for example, from the culture collection of the Bigelow Laboratory for Ocean Sciences, Center for Collection of Marine Phytoplankton (McKown Point, West Boothbay Harbor, Maine, 04575). Any of these diatoms can be grown using the methods and nutrients described in U.S. Patent No. 5,635,493 which is incorporated herein by reference. P-GlcNAc fibers can be obtained from diatomaceous cultures such as those described above through numerous different methods. In accordance with the Mechanical Strength method, p-GlcNAc fibers can be separated from diatomaceous cell bodies by subjecting the contents of the culture to an appropriate mechanical force. Said mechanical force may include, but is not limited to, a cutting force generated, for example, with a colloid mill, an ultrasonic device, or a bubble generator, or a shearing force generated, for example, by a mixer Waring. The resulting suspension of diatomaceous cell bodies and the p-GlcNAc fibers are then secreted. For example, the suspension can be subjected to a series of centrifugation steps that secrete the p-GlcNAc fibers from the cell bodies, providing a clear supernatant that has little visible flocculation material if it has some of said material. A fixed angle rotor, and a temperature of about 10 ° C are preferred for the centrifugation steps. The speed, duration and the total number of centrifugation steps required may vary according to, for example, the specific centrifugation rotor employed, but the determination of the values for such parameters will be apparent to a person with ordinary skill in the art. The p-GlcNAc fibers in the supernatant can then be concentrated using techniques well known to those skilled in the art. Such techniques may include, without limitation, fiing and suction devices. Finally, the concentrated p-GlcNAc fibers are washed, for example, with distilled-deionized water, HCl and ethanol, or other suitable solvents, preferably solvents such as alcohols in which both organic and inorganic materials are dissolved. An example that demonstrates the use. of this method for the purification of p-GlcNAc is presented in the Section of Example 6, infra. In accordance with the chemical / biological method, p-GlcNAc fibers are separated from diatom cell bodies by subjecting said bodies to chemical and / or biological agents. For example, you can treat crops of diatoms with a chemical agent capable of weakening the walls of diatomaceous cells, which causes the release of p-GlcNAc fibers without altering their structure. Said chemical agent may include, without limitation, hydrofluoric acid (HF). Alternatively, a mature diatomaceous culture can be treated with a biological agent that can alter a biological process and can be used to inhibit the synthesis of p-GlcNAc fibers, thereby releasing the fibers already present. For example, said agent may include, without limitation, polyoxin-D, an inhibitor of the enzyme N-acetylglucosaminyl-P-transferase. Cell bodies and fibers containing p-GlcNAc from diatomaceous cultures treated with a member of the chemical or biological agents described above are then secreted. For example, the contents of treated diatomaceous cultures can be allowed to settle in such a way that the contents of the cultures can form two distinct layers. The upper layer contains primarily the p-GlcNAc fibers, while the lower layer contains the bodies of the cells. The upper layer containing p-GlcNAc fibers can be removed with a siphon, leaving behind the settled cellular material of the lower layer. The layer containing: p-GlcNAc fibers removed by siphon can then be further purified in order to remove protein and other unwanted matter by treatment with a detergent that does not damage p-GlcNAc fibers. Said detergents may include, without limitation, sodium dodecyl sulfate (SDS). When an acid treatment is used, such as for example treatment with HF in order to separate the p-GlcNAc fibers from: the bodies of the diatomaceous cells, a step can be included for the dispersion of the fibers. Said step may include, without limitation, the use of mechanical force for the dispersion of the fibers, such as for example a step in which the fibers are subjected to the movements of an orbital stirrer. Alternatively, the acid treated suspension may, in an optional step, be neutralized before further purification by treatment with detergent. Said neutralization will change, in general, the pH of the suspension from about 1.8 to about 7.0, and may be accompanied, for example, by the addition of an appropriate volume of Tris ÍM (pH 8.0) or the addition of an appropriate volume of sodium hydroxide (NaOH). Neutralization, in general, provides pure p-GlcNAc fibers of substantially greater length than the other purification methods discussed herein. The purified p-GlcNAc fibers can then be concentrated using techniques well known to those skilled in the art, such as for example the use of a suction and filtration device. Finally, the p-GlcNac fibers are washed, in a series of steps with distilled-deionized water, HCl and ethanol, or other suitable solvents, preferably solvents such as alcohols, in which both organic and inorganic materials are dissolved. An example demonstrating the successful use of said purification method is presented in the example section 7, infra. While each of its methods for the purification of p-GlcNAc from microalgae, preferably diatoms, initial sources produce very pure, unadulterated, crystalline p-GlcNAc, each of the methods provides p-GlcNAc having specific and beneficial characteristics . For example, p-GlcNAc purified by the Mechanical Strength method produces a p-GlcNAc membrane that offers a superior substrate for the attachment of cells on p-GlcNAc. The chemical / biological method produces an average yield much higher than the average yield of p-GlcNAc obtained by the Mechanical Strength method. In addition, the variation of the acid treatment / neutralization of the chemical method / roduce extremely long p-GlcNAc fibers, some fibers exceeding 100μm, and containing molecules of the p-GlcNAc polymer of very high molecular weight, up to 20-30 million daltons . The electron micrographic structure of p-GlcNAc a employed in the compositions and methods of this invention, produced. using the variation of acid treatment / neutralization of the chemical / biological purification method is illustrated in Figure 3. The purification of the p-GlcNAc figures often results in the formation of fibrous membranes in accordance with that shown in Figure 3. 5.1.1. Derivation of p-GlcNAc The fully acetylated p-GlcNAc of the invention can be derived, employing various conditions and controlled procedures in a wide range of different compounds. See figure 4 for a diagram showing some of these compounds. Such derivative compounds may include, but are not limited to, partially or fully deacetylated p-GlcNAc, which has been modified through chemical and / or enzymatic means in accordance with what is described in further detail below. In accordance with a preferred embodiment of the invention, p-GlcNAc is a 100% deacety p-GlcNAc Lada. In addition, p-GlcNAc or its deacetylated derivative can be derived by being sulphated, phosphorylated, and / or nitrated. In addition, as explained in detail below, O-sulfonyl, N-acyl, O-alkyl, N-alkyl, deoxyhalogen, and N-alkylidene and N-arylidene and other derivatives can be prepared from the p- GlcNAc or p-GlcNAc deacetylated of the present invention. The deacetylated p-GlcNAc of the present invention can also be used for several organic salts and / or various metal chelates. In accordance with a preferred embodiment of the invention, one or more monosaccharide units of the p-GlcNAc can be deacetylated to form a species of poly-β-l- > Deacetylated 4-N-glucosamine. A kind of poly-ß-l- > 4-N-glucosamine wherein each of the monosaccharide units of the species poly-β-l- > 4-N-acetylglucosamine has been deacetylated, ie, a 100% deacetylated derivative, it will have a molecular weight of about 640,000 daltons to about 24 million daltons, with about 640,000 daltons being preferred to about 2.5 million daltons. A species with such a range of molecular weights represents a species having about 4,000 to about 150,000 glucosamine monosaccharides covalently linked in a β-1 -> configuration. 4. The p-GlcNAc may be deacetylated by treatment with a base to provide glucosamines with free amino groups. This hydrolysis process can be carried out with concentrated sodium hydroxide solutions or concentrated potassium hydroxide at elevated temperatures. See, for example, example section 8, infra. Alternatively, an enzymatic method employing a chitin deacetylase enzyme can be used for the deacylation of p GlcNAc. Said enzymatic process with deacetylase is well known to those skilled in the art and can be carried out in accordance with that indicated in U.S. Patent No. 5,219,749, which is incorporated herein by reference in its entirety. In addition, one or more inonosaccharide units of the p-GlcNAc of the invention can be derived in such a way that it contains at least one sulfate group or, alternatively, it can be phosphorylated or nitrated, in accordance with the illustration below: wherein R and / or R t instead of a hydrogen and / or R 2, instead of -COCH 3, may be a sulfate group (-SHO 3), a phosphate group (-P (.} OH) 2) or a nitrate group (-N02). Next, methods are described through which such p-GlcNAc derivatives can be prepared. Before carrying out these methods, it is useful to lyophilize first, freeze in liquid nitrogen and spray the initial material of p-GlcNAc.

Sulfated derivatives of p-GlcNAc can be generated by, for example, a two-step process. In the first step, O-carboxymethyl p-GlcNAc can be prepared from; p-GlcNAc and / or p-GlcNAc derivatives of the invention by, for example, the use of techniques such as those described by Tokura et al, (Tocura, S. Et al., 1983, Polym J. 15: 485) . Second, the sulphation step can be carried out for example with N, N-dimethyl-formamide-sulfur trioxide, in accordance with techniques well known to those skilled in the art, such as those described by Schweiger (Schweiger, RG, 1972). , Carbohydrate Res. 21219). The resulting product can be isolated as a sodium salt. Derivatives of phosphorylated p-GlcNAc can be prepared, for example, by using techniques well known to those skilled in the art such as the techniques described by Nishi et al. (Nishi, N. et al., 1986 in "" Chitin in Nature and Technology, Muzzarelli et al., Eds. Plenum Press, New York, pp. 297-299). In summary, a mixture of p-GlcNAc / methanesulfonic acid can be treated with phosphorus pentoxide (in a proportion of about 0.5 to 4.0 molar equivalents) with stirring, at a temperature of about 0 ° C to about 5 ° C. The treatment can last approximately 2 hours. The resultant product can then be precipitated and washed using standard techniques well known by the experts in the matter. For example, the sample can be precipitated with a solvent such as ether, centrifuged, washed with a solvent such as ether, acetone or methanol and dried. N-pivaled derivatives of p-GlcNAc can be prepared by using techniques well known to those skilled in the art; in the matter as for example the techniques described by Schorigin and Halt (Schorigin, R. And Halt, E., 1934, Chem Ver. 67: 1712). Briefly, p-GlcNAc and / or a p-GlcNAc derivative can be treated with concentrated nitric acid in order to form a stable nitrated product. One or more of the acarid monkey units of the p-GlcNAc of the invention may contain a sulfonyl group in accordance with that shown below: wherein R3 may be an alkyl, aryl, alkenyl or alkyne portion. Said derivative can be generated through well-known methods such as the known method in Kurita et al. (Kurita, K. et al., 1990, Polym, Prep [Am. Chem. Soc., Div. Polym. Chem.] 31; 624-625). In summary, an aqueous alkaline solution of p-GlcNAc can be reacted with a chloroform soot of tosyl chloride, and the reaction can then be allowed to proceed smoothly at low temperatures. One or more of the monosaccharides of the p-GlcNAc of the present invention or its deacetylated derivative may contain one or more O-acyl groups in accordance with the following: cppco 4 wherein R 4 and / or R 5 in place of hydrogen may be an alkyl portion, an alkenyl portion, or an alkynyl portion, and Rβ may be an alkyl portion, an alkenyl portion or an alkynyl portion. An example of such a derivative can be generated by well known methods such as the methods described by Ko ai (Ko ai, T. et al., 1986, in "Chitin in Nature and Technology", Muzzarelli et al., Ed > ., Plenum Press, New York, pp. 497-506). In summary, p-GlcNAc: can react with any of numerous chlorides; Suitable acyl derivatives in methanesulfonic acid to provide p-GlcNAc derivatives including, but not limited to, caproyl, capryl, lanoyl or bensoyl derivatives. One or more of the monosaccharides of the deacetylated p-GlcNAc of the invention may contain an N-acyl group, according to what is described below. wherein R7 may be an alkyl portion, an alkenyl portion, or an alkynyl portion. Said derivation can be obtained by the use of techniques well known to those skilled in the art, for example, the technique described in Hirano et al. (Hirano, S. Et al., 1976, Carbohylrate Research 47; 315-320). p-GlcNAc deacetalide is soluble in numerous aqueous solutions of organic acids. The addition of selected carboxylic anhydrides to such solutions containing p-GlcNAc in metalonic acetic acid aqueous, results in the formation of N-acyl derivatives p-GlcNAc. N-acyl p-GlcNAc is a preferred derivative for the production of controlled release drug delivery systems. One or more of the monosaccharides of the p-GlcNAc of the invention or of its deacetylated derivative may contain an O-alkyl group in accordance with that illustrated below: wherein Rβ may be an alkyl portion, an alkenyl portion or an alkynyl portion. Said derivation can be obtained by employing techniques well known to those skilled in the art. For example, the procedure described by Maresh et al. (Maresh, G. Et al., In "" Chitin and Chitosan, "Skjak-Braek, G. Et al., Eds., 1989, Elsevier Publishing Co., pages 389-395.) In summary, it can be dispersed -GlcNAc deacetylated in dimethoxyethane (DME) and can react with an excess of propylene oxide.The reaction period can be 24 hours, and the reaction is carried out in an autoclave at a temperature between 40 and 90 ° C. The mixture can then be diluted with water and filtered. The DME can be removed by distillation. Finally, the final product can be isolated through lyophilization. O-alkyl-p-GlcNAc and. its deacetylated derivative is also a preferred derivative for the production of controlled release drug delivery systems. One or cirias of the monosaccharide units of the p-GlcNAc of the invention may be an alkaline derivative in accordance with that illustrated below: Such a derivative can be obtained by the use of techniques well known to those skilled in the art. For example, a method such as the method described by Noguchi et al. (Noguchi, J, et al., 1969, Kogyo Kagaku 2; asshi 72 / 796-799). Briefly, p-GlcNAc can be impregnated, under vacuum, in NaOH (43%, preferably) for a period of about 2 hours at a temperature of about 0 ° C. The excess NaOH can then be removed, for example, by centrifugation in a basket centrifuge or by mechanical braiding.

One or more of the monosaccharide units of the deacetylated derivative of p-GlcNAc may contain an N-alkyl group, as illustrated below: wherein Rg can be an alkyl portion, an alkenyl portion or an alkynyl portion. Said derivation can be obtained by using, for example, a method such as the procedure of Maresh et al. (Maresh, G. et al., In "Chitin and Chitosan" Skjak-Brack, G. et al., Eds., 1989, Elsevier Publishing Co., pages 389-395), in accordance with what is described above, for production of N-alkyl p-GlcNAc derivatives. One or more of the monosaccharide units of the deacetylated derivative of the p-GlcNAc of the invention may contain at least one deoxyhalogen derivative, as illustrated below: H fvH2 wherein Rio can be F, Cl, Br or I, with preference I. This derivative can be obtained by using techniques well known to those skilled in the art. For example, a method such as the procedure described by Kurita et al. (Kurita, K, et al., 1990, Poly. Prep. [Am. Chem. Soc. Div. Polym. Chem.] 31 / 624-625). Briefly, a tosylated p-GlcNAc is reacted with a sodium halide in dimethyl sulfoxide to provide a deoxyhalogen derivative. The tosylation of p-GlcNAc can be effected by the reaction of an aqueous alkaline p-GlcNAc solution with a chloroform solution of tosyl chloride. Said reaction can be carried out gently at low temperatures. One or more of the monosaccharide units of the deacetylated derivative of the p-GlcNAc of the invention can form a salt, in accordance with that shown below: CHjX wherein R ^ can be an alkyl portion, an alkenyl portion, or an alkynyl portion. Said derivation can be obtained by using well-known techniques by experts in the field. For example, a procedure for example the procedure described by Austin and Sennett (Austin PR and Sennett, S., in "Chitin in Nature and Technology", 1986, Muzzarello, RAA et al., Eds. Plenum Press, pages 279 -286) can be used. In summary, deacetylated p-GlcNAc can be suspended in an organic medium such as for example ethyl acetate or isopropanol, to which an appropriate organic acid can be added, such as for example formic acid, acetic acid, glycolic acid or lactic acid. The mixture can be allowed to sit for a certain period of time (from one to three hours, for example). The reaction temperature and drying may vary from about 12 ° C to about 35 ° C, with from about 20 ° to about 25 ° C being preferred. The salts can be separated by filtration, washed with fresh medium, and the residual medium can be evaporated. One or more of the monosaccharide units of the deacetylated derivative of the p-GlcNAc of the invention can form a metal chelate according to that shown below. wherein R12 can be a metal ion, especially an ion of the transition metals, and X the dative bond, established by the nitrogen electrons present in the amino and substituted amino groups present in the deacetylated p-GlcNAc. One or more of the monosaccharide units of the deacetylated derivative of the p-GlcNAc of the invention may contain an N-alkylidene group or an N-arylidene group, as illustrated below: wherein R13 may be an alkyl portion, an alkenyl portion, an alkynyl portion, or an aryl portion. Said derivation may be obtained by the use of techniques well known to those skilled in the art. For example, a method such as the procedure described by Hirano et al., (Eirano, S. et al., 1981, J. Biomed, Mat. Res. 15: 903-911) may be employed. Briefly, a N-substituted reaction of deacetylated p-GlcNAc can be carried out with carbonylic anhydrides and / or arylaldehydes to provide acyl and / or arylidene derivatives. In addition, p-GlcNAc or its deacetylated derivative can be subjected to controlled hydrolysis conditions that provide groups of molecules that have discrete uniform molecular weights and other physical characteristics. Said hydrolysis conditions, may include, for example, treatment with the enzyme lysozyme. p-GlcNAc may be exposed to lysozyme for varying periods of time in order to control the magnitude of the hydrolysis. In addition, the rate of hydrolysis can be controlled depending on the magnitude with which p-GlcNAc in the process of treatment with lysozyme has been deacetylated. The deactivation conditions can be in accordance with what has been described above. The more deacetylated a p-GlcNAc molecule, between about 20% and about 90% deacetylated, the greater the hydrolysis of a molecule at a given time. Changes in room to physical characteristics, in addition to the decrease in molecular weight, can be caused by hydrolysis and / or deacetylation treatment. Extensive hydrolysis causes the liquefaction of p-GlcNAc. In addition, thermal denaturation can function to modify the crystal structure of p-GlcNAc. Said modification of the crystalline structure of p-GlcNAc product can have a beneficial effect, for example, on the reactivity of p-GlcNAc. In addition, hybrids comprising p-GlcNAc and / or p-GlcNAc derivatives can be formed. Such hybrids can contain any of numerous natural and / or synthetic materials, in addition to p-GlcNAc and / or p-GlcNAc derivatives. For example, p-GlcNAc hybrids and / or p-GlcNAc derivatives plus one or more extracellular matrix (ECM) components can be formed. Such EMC components may include, but are not limited to, collagen, fibronectin, glycosaminoglycans, and / or peptidoglycans. P-GlcNAc hybrids and / or p-GlcNAc derivatives can also be formed together with one or more synthetic materials such as polyethylene. Such p-GlcNAc / polyethylene hybrids can be formed by the thermal bonding of the hybrid components for example by autoclaving. The p-GlcNAc derivatives for use in the claimed invention are derivatives of deacetylated p-GlcNAc salts such as p-GlcNAc lactate derivatives, especially derived from p-GlcNAc lactose gel. As used herein, the term "p-GlcNAc lactate" means that the lactic acid moiety is functionally fixed on a partially or fully deacetylated p-GlcNAc Such p-GlcNAc lactate derivatives can be obtained in accordance with described above (for example by derivatization with lactic acid) and formulated in the form of a gel using propylene glycol and water, according to what is described in example section 10, infra P-GlcNAc lactate derivatives can be produced which have high and low viscosities, which allows the ability to adapt the p-GlcNAc to the specific indication of interest. For example, you can > It is advantageous to employ a p-GlcNAc having a lower viscosity for administration through a syringe or through spraying. In accordance with what is described in more detail in section 5.3, infra, p-GlcNAc and / or its derivatives in accordance with what has been described above, can be further derivatized through covalent or non-covalent attachment or combination with molecules or drugs of interest bales as endothelin antagonists. 5.1.2 Reformulations of p-GlcNAc p-GlcNAc, their deacetylated derivatives and / or their derivatives, such as those described above, to be used in the compositions of the invention, can be dissolved and subsequently reformulated in various forms and configurations. A solution of p-GlcNAc can be achieved by treatment with dimethylacetamide (DMA) / lithium chloride. P-GlcNAc can be easily dissolved by stirring in a solution of DMA containing 5% LiCl (by weight of the DMA). Water-soluble p-GlcNAc derivatives, such as p-GlcNAc salts, for example, carboxymethyl or lactate derivatives, may be dissolved in water. p-GlcNAc that has been deacetylated by at least about 75% can be introduced into the solution, for example in a slightly acidic solution, such as for example 1% acetic acid. Derivatives of p-GlcNAc that are insoluble in water can be placed in solution in organic solvents. The derivation of p-GlcNAc in DMA: LiCl with phenyl isocyanates can be used to produce carbanylates. In addition, the derivatization of p-GlcNAc in DMA: LiCl with p-sulfonylchloride of toluene can be used to produce p-sulfonate of toluene. The p-GlcNAc, its deacetylated derivatives, and / or its derivatives in solution can then be precipitated and reformulated in forms including, but not limited to, mats, chains, microspheres, microbeads, membranes, fibers, powders, sponges and gels. In addition, ultra-thin uniform membranes (i.e., of a thickness less than about 1 miera) can be formulated. In addition, pharmaceutical formulations such as pills, tablets and capsules can be prepared. Such reformulations can be achieved, for example, by taking advantage of the fact that pure p-GlcNAc is insoluble in solutions such as water and alcohol, preferably ethanol. The introduction, by conventional means, such as by means of; Injection, for example, of the DMA / LiCl mixture containing p-GlcNAc in said water or alcohol solution, preferably ethanol, will cause reprecipitation and consequently reformulation of the dissolved p-GlcNAc. The Reformulation of a p-GlcNAc membrane in a fibrous material is demonstrated in the example section 9, infra. In the case of water-soluble p-GlcNAc derivatives, reformulations can be achieved by reprecipitation in organic solvents such as, for example, ethyl acetate or isopropanol, reformulations of p-GlcNAc which has been deacetylated at least in approximately 75% can be achieved by reprecipitation in an alkaline solution. Water-insoluble p-GlcNAc derivatives can be reformulated by reprecipitation in aqueous solutions, such as water. P-GlcNAc membranes and three-dimensional p-GlcNAc matrices can be produced through methods that provide for the formation of controlled average pore sizes within either the membranes or the matrices. The pore size can be controlled in membranes and matrices by varying the amount of p-GlcNAc material that is used, or by the addition of certain solvents such as methanol, or ethanol, with ethanol being preferred, in specific quantities, which is located within a range of about 5% to about 40% before the formation of membranes and / or matrices. In general, the higher the percentage of solvent, the smaller the average pore size formed. In accordance with a preferred reformulation of the invention, a p-GlcNAc lactate derivative is formulated in a gel according to what is described in detail in the section example 10, infra. 5.2 Endothelin antagonists of the compositions of the invention Endothelin antagonists to be employed in the compositions and methods of the invention include, but are not limited to, endothelin antagonists based on peptides, endothelin antagonists not based on peptides, endothelin antagonists specific for ETA, specific for ETB or non-specific. Examples of endothelin receptor antagonists based on peptides useful in the compositions and methods of the invention include BQ-123 (Cyclo (-D-Trp-D-Asp-L-Pro-D-Val-L-Leu-), BQ -153, B?) - 238, BQ-485, BQ-160, BQ-788, BQ-928, TAK-044, FR139317 (Perhydroazepin-1-ylcarbonyl-L-leucyl- (1-methyl) -D-triptofil - [3- (2-pyridyl)] -D-alanine), RES-701-1 (Novabicchem), PD 142893 (Acetyl- (3, 3-diphenyl-D-alanine) -L-Leu-L-Asp-L-Ile-L-Ile-L-Trp), PD 145065, PD 170687, Ac- DBhgl6-Leu-Asp-Ile, IRL-1038 ([Cisll-Cisl5] -endotelin-1 (11-21)), the pentapeptide GRGDS, and ET-1 [Dpr] -Asp 15]. Many of these peptides can be obtained commercially, for example at the American Peptide Company, Sunnyvale CA or Calbiochem-Novabiochem Company, San Diego CA. Examples of non-peptide-based endothelin receptor antagonists for use in the compositions and methods of the present invention include Ro 61-0612, Ro 61-1790, Ro. 42-2005, Ro 46-2005, Ro 46-8443, Ro 47-0203 (also known in the art as bosentan), PD 155080, PD 156707, SB 209670, SB 217242, L-744,453, L-749,329, L- 754,142, CGS 27830, BMS 182874, LU 135252, S-1039, mA386, A-127722, TBC11251, Nz-arg-3- (isoxadylsulfamethyl) -2-thiophenecarboxamide and EQ 123. See, for example, Webb er al., supra and Ohlstein et al., supra, for structures of many of these known antagonists of endothelin. Non-peptide-based endothelin antagonists may be preferred according to this invention since they exhibit more favorable pharmacokinetic properties than peptide-based antagonists, eg, greater metabolic stability and greater bioavailability and oral activity. According to a preferred embodiment of the invention, the endothelin antagonist used is Rodl according to that illustrated in Figure I, supra. 5.3 Preferred Formulations of the Compositions of the Invention According to a preferred embodiment of the invention, an endothelin antagonist according to that described above ("EA") is functionally fixed covalently or non-covalently or is combined with the p-GlcNAc, or one or more derivatives or reformulations thereof, in accordance with that described above. In accordance with a In one embodiment, at least one type of endothelin antagonist is combined or mixed, covalently, non-covalently or otherwise with a deacetylated p-GlcNAc. Other a.ntitumor agents that can be employed in combination with the EA / p-GlcNAc compositions of the invention are discussed supra. The endothelin antagonist or other antitumor agent can be covalently linked to the exposed primary amines of the deacetylated p-GlcNAc, for example, through chemical bonding using bifunctional crosslinking reagents that act as chemical spacers of specific length. Such techniques are well known to those skilled in the art and may resemble, for example, the methods of Davis and Preston (Davis, M. and Preston, J.F. 1981, Anal. Biochem. 116: 404-407) and Staros et al. (Staros, J.V. et al., 1986, Anal. Biochem. 156: 220-222). For example, in the case of peptide-based compounds, carboxylic residues in the peptide to be linked to the deacetylated or partially deacetylated p-GlcNAc can be activated and then cross-linked with the p-GlcNAc. The activation can be achieved, for example by the addition of a solution such as carbodiimide EDC (l-ethyl-3- (3-dimethylaminopropyl) carbodiimide) to a solution of peptide in a phosphate buffer. Preferably, this solution also contains a reagent such as a sulfo-NHS (N-hydroxysulfosuccionimide) to increase the coupling. The activated peptide can be crosslinked on the deacetylated p-GlcNAc by mixing in a high pH buffer, such as for example carbonate buffer (pH 9.0 - 9.2) The biological activity of the bound molecule can be maintained by varying the length of the the linker molecule (for example, the bifunctional crosslinking compound) which is used to bind the molecule on the p-GlcNAc. An appropriate linker length for a given molecule to be linked that does not alter the biological activity of the linked molecule can be determined routinely. For example, the biological activity (eg, a therapeutically effective level of biological activity) of a molecule that has been linked through a linker of a given length can be proved by the use of well-known assays specific for the molecule given in link process. In addition, with the object of maintaining the biological activity of the molecule that is being held, it is necessary to use a linker that can be dissociated through an appropriate enzyme that occurs in nature to release the bound molecule. Assays commonly employed by those skilled in the art can be used to test the retention of the biological activity of a particular bound molecule to ensure that it is maintains an acceptable level of activity (for example, an activity of therapeutically effective level). Alternatively, peptide-based or non-peptide-based endothelin antagonists, alone or in combination with other antitumor agents, can be mixed or non-covalently linked to p-GlcNAc and / or its derivatives to form the compositions of the invention. invention, employing techniques well known to those skilled in the art. For example, a molecule or molecules of choice, for example, an endothelin antagonist, can be mixed with suspensions of p-GlcNAc, with a deacetylated or partially deacetylated p-GlcNAc solution, with a deacetylated p-GlcNAc salt solution. or partially deacetylated, for example, with a solution of p-GlcNAc lactate (partial or total deacetylated reagent) or with any solution derived from p-GlcNAc, the mixtures may optionally be lyophilized. The molecules can be non-covalently bound to the p-GlcNAc matrices after lyophilization, probably through hydrophobic, electrostatic or other non-covalent interactions. Such formulations of p-GlcNAc are very easy to produce. In addition, such formulations can be obtained effectively with a wide variety of molecules that have a broad spectrum of physical characteristics and water solubility properties, found within from a range of the most hydrobocic to the most hydrophilic. When linked: the molecule or molecules, assays commonly employed by those skilled in the art to test the activity of the particular molecule or molecules linked non-covalently can be used to ensure that an acceptable level of activity is achieved (e.g. , a therapeutically effective activity) with the linked molecule. In addition, endothelin antagonists alone or in combination with other antitumor agents may be encapsulated in p-GlcNAc using methods known in the art. For example, a method to achieve encapsulation may involve the procedure presented by Hwang et al.

(Hwang, C. et al., In Muzzarelli, R. Et al., Eds., 1985, "Chitin: 'n Nature and Technology," Plenum Press, pages 389-396) which is incorporated herein by reference in its entirety. . The encapsulation can also be achieved, for example, by following a modification of the acid treatment / neutralization variation of the chemical / biological purification method presented above. Instead of raising the pH of the p-GlcNAc solution to an approximately neutral pH range (ie, approximately if it is 7.4), a basic pH environment can be created by raising the pH to approximately 9.0 after purification of p-GlcNAc. At a more basic pH, the structure of p-GlcNAc, or a derivative of it, takes a more three-dimensional or "open" configuration. When the pH is lowered, the configuration of the molecule returns to a more compact, "closed" configuration. Thus, a drug compound of interest, such as for example an endothelin antagonist, can be added to a solution of p-GlcNAc at a high pH, then the pH of the p-GlcNAc / drug suspension can be lowered, "trapping" in this way or encapsulating the drug of interest within a p-GlcNAc matrix. By encapsulating the molecule, assays commonly employed by those skilled in the art may be employed to test the activity of the particular molecule or molecules encapsulated, thereby ensuring that an acceptable level of biological activity (eg, a therapeutically effective activity) is maintained by part of the encapsulated molecule. An example of the preparation of an EA / p-GlcNAc composition of: the invention is presented in Example section 10, infra, where an endothelin antagonist is mixed with a p-GlcNAc lactate gel. Alternatively, it is possible to prepare an EA composition (without a p-GlcNAc accompanying it), for example, by dissolving the endothelin antagonist in PBS, HBSS or in accordance with that described in the manufacturer's instructions and adjusting the solution to the desired concentration.

Compositions of the invention, including EA / p-GlcNAc compositions can be formulated for administration as pharmaceutical compositions, for example, by inhalation or insufflation (either through the mouth or nose), or through oral, oral, parenteral or rectal administration. In accordance with a preferred embodiment, the EA / p-GlcNAc composition of the present invention is administered by injection in the form of a gel in accordance with that described in Example section 10, infra. In the embodiment of the invention, wherein the pharmaceutical composition comprises the administration of an endothelin antagonist, for example, an endothelin antagonist, for example a non-peptide endothelin antagonist, such as pyrimidylsulfonamide, for the treatment of a proliferative disease such as cancer, the composition may comprise a therapeutically effective amount of the endothelin antagonist in combination with a pharmaceutically acceptable carrier. For oral administration, the pharmaceutical compositions can take the form as, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients or vehicles such as binders (for example, pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (e.g., para starch or sodium starch glycolate); or wetting agents (for example, lauryl sulfate sodium). The p-GlcNAc can be used in place of the excipients, vehicles and fillers or in addition to said excipients, vehicles and fillers. Tablets can be coated using p-GlcNAc using methods well known in the art. Liquid preparations for oral administration can take the form, for example, of solutions, syrups or suspensions, or they can have the presentation of a dry product for constitution with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoate or sorbic acid). The preparations may also contain buffers, salts, flavors, colorants and sweeteners as appropriate. . 4 Uses of the compositions and methods of the invention Biomedical uses of the compositions of the invention include their use as drug delivery systems for endothelin antagonists as well as other therapeutic agents such as other antitumor agents. The p-GlcNAc-containing formulations of the invention offer added benefits compared to known pharmacological formulations, including, for example, greater effectiveness, lower toxicity and greater bioavailability. In fact, there are numerous advantages in using the drug delivery systems based on p-GlcNAc of the invention. For example, traditional administration of drug by injection is commonly used with proteins and many other drugs. However, repeated doses provoke oscillating blood drug concentrations and affect patient comfort and compliance. Oral administration can be helpful since it allows a more varied load of the drug to be released and is less uncomfortable for the patient. However, proteins and other compounds are denatured and degraded in the stomach. An improved oral administration, however, is achieved through compositions containing p-GlcNAc of the invention providing a protective environment for the drug once administered. For example, p-GlcNAc protects a peptide-based endothelin antonist against the acidic and enzymatic environment of the stomach. The p-GlcNAc system releases the compound through diffusion and / or degradation of the encapsulation once it reaches the region of the intestine where it is effectively absorbed into the bloodstream. These p-GlcNAc systems of the invention can be used, for example, to administer proteins as well as many other compounds. Liposomes coated with p-GlcNAc derivatives or alginate encapsulations of p-GlcNAc derivatives are preferred for these oral administration methods. In addition, by introducing the compositions of the present invention into a patient, the p-GlcNAc degrades with the passage of time, such that linked or adjunct compounds are gradually released into the patient's bloodstream, thus providing a method for administration of controlled slow release of the drug. Deacetylated or partially deacetylated p-GlcNAc species can be produced which have a predictable rate of biodegradability. For example, the percentage of deacetylation affects the rate at which the p-GlcNAc species degrade. In general, the greater the percentage of deacetylation, the faster the rate of biodegradability and resorption. Thus, the degree of biodegradability of p-GlcNAc and the in vivo rate of resorption can be controlled during the production of p-GlcNAc.

GlcNAc. P-GlcNAc materials having such controllable rates of biodegradability can be formulated into membranes, gels, sponges, microspheres, fibers and the like. In accordance with a preferred embodiment of the invention, a deacetylated or partially deacetylated p-GlcNAc having a predictable rate of biodegradability can be employed. Compositions of p-GlcNAc / drug of the invention can be administered in a patient through several routes using standard procedures well known to those skilled in the art. For example, said administration may be an oral, nasal, intravenous, subcutaneous, intradermal, transdermal, intramuscular or intraperitoneal, site-specific administration. In terms of site-specific administration, methods of administration may include, but are not limited to, injection, implant, arthroscopic, laparoscopic, or similar devices. Membranes of p-GlcNAc and / or gels as well as microspheres and sponges are preferred for such site-specific administration methods. As noted supra, the p-GlcNAc of the compositions of the invention will be formulated into membranes, gels, sponges, microspheres, fibers and the like. These products of p-GlcNAc adhere and mold on the tissues, both tissues soft as hard tissues, in the human body, without the need for suturing. The p-GlcNAc materials can, for example, be applied during a general or minimally invasive surgical procedure, such as a laparoscopic surgical procedure. In accordance with a preferred embodiment of the invention, p-GlcNAc is in the form of a gel wherein the endothelin antagonist and / or other antitumor agent is dissolved or otherwise incorporated. Gels based on p-GlcNAc and membranes have various applications as therapeutic pharmacological delivery systems, for example, to provide a site-specific slow release administration directly on a tumor or on the region evacuated by a tumor after a surgical intervention. Such an immobilized slow release composition can act as an important initial defensive procedure after a surgical intervention. Furthermore, such antitumor drug delivery systems can be especially useful for the treatment of tumors totally or partially inaccessible to a surgical intervention, as for example in the case of certain brain tumors. The EA / p-GlcNAc compositions of the present invention are therefore useful as therapeutic drug delivery systems for the treatment of cancer and other proliferative diseases. These compositions may further include other antitumor agents which can be fixed on the p-GlcNAc of the invention or encapsulated within said p-GlcNAc in order to offer a synergistic effect. Such antitumor agents are well known to those skilled in the art, and include, but are not limited to, the following specific categories and compounds: alkylating agents, antimetabolite agents, antitumor antibiotics, vinca alkaloid and epidofilotoxin agents, nitrosoureas, enzymes, Synthetic drugs, biological therapeutic hormones and research. Such alkylating agents include, but are not limited to, nitrogen mustard, chlorambucil, cyclophosphamide, ifosfamide, melphalan, triptepa and busulfan. Antimetabolites may include, but are not limited to, methotrexcLto, 5-fluorouracil, cytosine arabinoside (ara-C), 5-azacytidine, 6-mercaptopurine, 6-thioguanine, and fludarabine phosphate. Antitumor antibiotics may include, but are not limited to doxorubicin, daunorubicin, dactinomycin, bleomycin, mitomycin C, plicamycin, idarubicin, and mitoxantrone. Vinca alkaloids and epipodophyllotoxins may include, but are not limited to, vincristine, vinblastine, vindesine, etoposide, and teniposide. Nitrosoureas include carmustine, lomustine, semustine, and streptococcus. Enzymes can include, without limitation L- asparagine. Synthetics may include, but are not limited to, dacrabazine, hexamethylmelamine, hydroxyurea, mitotane procabazine, cisplatin, and carboplatin. Hormone therapeutic agents may include, but are not limited to, corticosteroids (cortisone acetate, hydrocortisone, prednisone, prednisolone, methyl prednisolone, and dexairtuthisone), estrogens, (diethylbysterol, estradiol, esterified estrogens, conjugated estrogens, chlorothiasnene), progestins (medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate), antiestrogen (tamoxifen), aromastase inhibitors (aminoglutethimide), androgens (testosterone propionate, methyltestosterone, fluoxymesterone, testolactone), antiandrogens (flutamide), analogues of LHRH (leuprolide acetate) and endocrine for prostate cancer (Ketoconazole). Biological agents may include, but are not limited to, interferons, interleukins, tumor necrosis factor, and biological response modifiers. Research drugs may include, but are not limited to, alkylating agents such as Nimustine AZQ, BZQ, cyclodison, DADAG, CB10-227, CY233, DABIS maleate, EDMN, Fotemustine, Hepsulfam, Hexamethylmelamine, Mafosamide, MDMS, PCNU, Spiromustine, TA-077, TCNU and Temozolomide; antimetabolites, such as acivicin, azacitidine, 5-aza- deoxycytidine, A-TDA, Benzylidenglucose, Carbetimer, CB3717, Deazaguanine mesylate, DODOX, Doxifluoridine, DUP-785, 10-EDAM, Fazarabine, Fludarabine, MZPES, MMPR, PALA, HRCT, TMQ, TNC-P and Piritrexim; antitumor antibodies, such as AMPAS, BWA770U, BWA773U, BWA502U, Amonafide, m-AMSA, CI-921, Datelite, Mitonafido, Piroxantrone, Aclarubicin, Citorodin, Epirubicin, Esorubicin, Idarubicin, Iodine-doxorubicin, Marcellomycin, Menaril, Morpholino anthracyclines , Pirarubicin, and SM-5887; microtubule spindle inhibitors, such as, for example, Amfetinila, Navelbina and Taxol; alkyl-lysophospholipids, such as BM41-440, ET-18-OCH3, and Hexacylphosphocholine; metal compounds such as Gallium Nitrate, CL286558, CL287110, Cycloplatam, DW2114R, NK121, Iproplatin, Oxaliplatin, Spiroplatin, Spirogermanium, and Titanium compounds; as well as novel compounds such as, for example, glycinate of Aphidocylin, Ambazone, BSO, Caracemide, DSG, Didemnin, B, DMFO, Elsamycin, Esperta-razin, Flavone acetic acid, HMBA, HHT, ICRF-187, Iododeoxyuridine, Ipomeanol, Liblo icin , Lonidamine, LY186641, MAP, MTQ, Merabarone, SK &F104864, Suramine, Talisomycin, Teniposide, THU and WR2721; and Toremifene, Trilosano and zindoxifene. Antitumor drugs that are radiation enhancers are preferred for example in the case of the prescription of a radiation therapy treatment either in lieu of a surgical intervention or after a surgical intervention. Examples of such drugs include, for example, the chemotherapeutic agents 5'-fluorouracil, mitomycin, cisplatin and its derivatives, taxol, doxorubicin, actinomycin, bleomycins, daunomycins, and metamycins. Additional synergistic effects can be obtained by using EA / GlcNAc compositions of the invention in combination with two or more other antitumor agents such as thioguanine combined with cytosine arabinoside (ara-C) for the improved treatment of acute non-lymphocytic leukemia, tamoxifen with cisplatin for cancer of breast, and prostaglandins with cisplatin for breast and prostate cancer. Many other synergistic combinations of anti-cancer drugs, known to those skilled in the art, can be employed with the EA / p-GlcNAc and EA / p-Glc ^ JAc derivatives of the invention. In addition, the use of the p-GlcNAc-containing compositions of the invention is desirable since the p-GlcNAc polymer has chemical properties and characteristics that make it possible to formulate and administer some drugs that have been difficult to formulate and administer to the patient. date. For example, taxol, a microtubule spindle inhibitor drug used to treat breast cancer, is hydrophobic and requires the addition of polyoxyethylated castor oil in order to solubilize it as an infusion. liquid for intravenous administration. The hydrophobic nature of taxol makes this drug an ideal compound for its formulation with p-GlcNAc polymer materials for topical administration of controlled release. U.S. Patent No. 5,635,493 in Section 23 which is incorporated herein by reference, presents such a formulation of p-GlcNAc / taxol. Additional objectives for p-GlcNAc antitumor systems include, but are not limited to, skin, gastrointestinal tract, pancreatic, lung, breast, urinary tract, and uterine tumors, as well as kaposi sarcomas related to HIV. Due to the fact that the p-GlcNAc materials of the invention are inherently immunoneutral, insofar as they do not elicit an immune response in humans, such p-GlcNAc devices, in accordance with what is described above, comprises membranes of p-GlcNAc, 3D porous matrices and / or gels containing immobilized drugs can deliver such drugs in such a way that there is no immune response. Certain additional materials such as natural alginates and synthetic polymers can be used in some cases to construct such devices in combination with the p-GlcNAc material. For example, a polymeric prolonged-release drug delivery system can be manufactured in a manner similar to that suggested by A. Polk (Polk, A. et al., 1994, J.

Pharmaceutical Sciences, 83 (2): 178-185). In a process of this type, deacetylated p-GlcNAc reacts with sodium alginate in the presence of calcium chloride to form microcapsules containing the drug to be administered and released under appropriate conditions and for a certain period of time. The therapeutically effective doses of any of the drugs or agents described above, in combination with the p-GlcNAc-based systems described herein can be determined routinely using techniques well known to those skilled in the art. A "therapeutically effective" dose refers to the amount of the compound sufficient to result in an improvement of the symptoms of the processes and / or diseases described above. The toxicity and therapeutic efficacy of the drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population). The ratio between the toxic effects and the therapeutic effects is what is known as the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compounds that exhibit high therapeutic indices are preferred compounds. While compounds that have side effects can be used Toxic, care should be taken to design a delivery system that directs such compounds to the site of the affected tissue in order to minimize potential damage to uninfected cells, and consequently in order to reduce side effects. The data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED5o with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound employed in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a range of circulating plasma concentration that IC50 inclines (ie, the concentration of the test compound that achieves maximum inhibition of symptoms I in accordance with that determined in culture. This information can be used to more accurately determine useful doses in humans.The levels in the plasma can be measured, for example, by high-performance liquid chromatography. with a preferred embodiment, the dose range of the endothelin antagonists employed in the compositions of the invention is from about 1 mg / kg to about 100 mg / kg. In addition, the doses of many of the antitumor drugs listed above are well known to those skilled in the art and can be easily found in reference documents such as PHYSICIANS DESK REFERENCE, Medical Economics Data Publishers; REMINGTON 'S PHARMACEUTICALS SCIENCES, Mack Publishing Co.; GOODMAN & GILMAN, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, McGraw Hill PubLishers, THE CHEMOTHERAPY SOURCE BOOK, Williams and Wilkens Publishers, online services such as Cancer Lit®, Database of the National Cancer Institute of the United States of America, as well as reports on pharmacological studies such as "A MultiCenter Randomized Trial of Trial of Two Doses of Taxol" Nabholtz, JM, Gelmon, K., Bontenbal, M. et al. Medical Education Services Monograph - 1994 Bristol-Myers Squibb Company Publication; "Randomized Trial of Trial of Two Doses of Taxol in Metastatic Breast Cancer: An Interim Analysis" Nabholtz, J.M., Gelmon, K., Bontenba .., M., et al. 1993, Proc. Am. Clin. Oncol., 12:60. Summary 42. The dose ranges for antitumor drugs in the compositions of the invention may be smaller, equal or greater than the typical daily doses prescribed for the systemic treatment of patients. For example, dosages of 5'-FU equivalent to 50% of the standard dosages used to treat colorectal cancer with 5'-FU in humans (300-450 mg / m2 iv daily for 5 days) resulted in a reduction of 80- 90% in terms of the volume of ectopic HT29 colon cancer tumor implants in scid mice. The use of the p-GlcNAc membrane as a drug delivery matrix for the administration of 5 '-FU reduced the dosage required to dramatically reduce the volume of the tumor: by 50% compared to the control animals treated in ways intravenous Details regarding these data can be found in the example section 21 of US Patent 5,635,493, which is incorporated herein by reference. In cases where higher doses are required, higher doses may be tolerated to the extent that the drugs are delivered locally at the site of a tumor, and therefore, other tissues, including blood cells, are not easily exposed. to the drugs. Certain antitumor agents are vesicants, including dactinomycin, daunomycin, doxorubicin, estramustine, mechlorethamine, mitomycin C, vinblastine, vincristine, and vindesine; while these antitumor drugs are irritating, including carmustine, decarbazine, etoposide, mitrimycin, strepto: ocin, and teniposide. Vesicants and irritants cause negative side effects including extravasation and irritation of tissues with pain, redness, swelling and other symptoms. In addition, tissue necrosis of some of these side effects may result. The p-GlcNAc membrane and gel materials of the compositions of the invention employed for topical controlled release of antitumor drugs have wound healing properties. Normal tissues in contact with vesicant or irritant antitumor drugs administered through the p-GlcNAc membrane formulations and gel of the invention therefore are not easily damaged and will present a faster healing due to the curative effects of the p-GlcNAc component of the compositions containing p-GlcNAc of the invention. 6. EXAMPLE: PURIFICATION OF P-GLCNAC USING THE MECHANICAL FORCE PURIFICATION METHOD In this section, p-GlcNAc was purified using the Mechanical Strength technique described in section 5.1, supra. 6.1 MATERIALS AND METHODS / RESULTS Diatom culture conditions: The Thalassiosira fluviatilis diatom species was cultivated in culture in accordance with the procedures described in US Pat. No. 5,635,493, which is incorporated herein by reference. reference. SEM procedures: The SEM techniques used here were the following: a Zeiss 962 instrument was used with an acceleration voltage of 10 kV, and a working distance of 15 mm. Polaroid type 55 p / n (u4) was used in several amplifications, as indicated. Sample coating: carbon coating (100a) & 100th apud. (a) Sample preparation: For primary fixation, the growing culture medium was replaced with 2% glutaraldehyde and Eagle DMEM without serum. Several changes were made to ensure a complete transition of the growth medium to the fixative. The fixation was continued for 0.5 hours at room temperature. Cover strips were transferred to fresh flasks containing 2% glutaraldehyde in Na 0 cacodylate at a pH of 7.2 with 0. 5 M sucrose and added for an additional 1.5 hours at room temperature. Purification procedure of p-GlcNAc: p-GlcNAc was purified from the diatomaceous culture by using the mechanical force technique described in section 5.1, supra. Specifically, the p-GlcNAc fibers were separated from the bodies of diatomaceous cells by subjecting the contents of the culture to three short pulses of high speed mixing motion in a Waring blender. The total time of these three impulses was about one second. The resulting suspension was centrifuged at 3500 revolutions per minute in a Sorvall GS-4 fixed-angle rotor for 20 minutes at a temperature of about 10 ° C. The supernatant was decanted and centrifuged again, this time at 4000 revolutions per minute. , in a fixed-angle rotor Sorvall GS-4 for 20 minutes at a temperature of approximately 10 ° C. Again, the supernatant was decanted and centrifuged at 4000 revolutions per minute at 10 ° C. The final supernatant of the third centrifugation was of course, with few phylocles, if any visible floating in the liquid. The clear supernatant was decanted in a Buchner filtration unit equipped with a polyurethane sulfone filter membrane Supor-800, with pore size of 0.8 μm (Gelman, Inc.), suction was then applied and the liquid was filtered from the suspension of fibers, allowing the fibers to be collected in the membrane. The collected fibers were washed with one liter of deionized H20, distilled at a temperature of 70 ° C. When almost all of the water had been drained, the fibers were washed, with suction, with one liter of 1 N HCl at a temperature of 70 ° C. When most of the acid solution had been drained, the fibers were washed with one liter of deionized H20, distilled at a temperature of 70 ° C, using suction. When most of the water in The wash had been drained, the fibers were washed with one liter of 95% ethanol at room temperature, and vacuum was applied. The filter membrane where the white fiber membrane had been collected was then removed from the filtration unit and the membrane and its membrane support were dried in a drying oven at a temperature of 58 ° C for 20 minutes, after which the membrane and its support were placed in a drying device for 16 hours. After this purification procedure, the yield of p-GlcNAc from the 1000 ml culture was 6.85 milligrams per liter of diatom culture. 7. EXAMPLE: PURIFICATION OF P-GLCNAC USING THE BIOLOGICAL / CHEMICAL PURIFICATION METHOD In this section, p-GlcNAc was purified using two of the chemical / biological techniques described in section 5.1, supra. In summary, p-GlcNAc was purified through treatment with HF, in one case, and through acid treatment / neutralization in the second case. 7.1. MATERIALS AND METHODS / RESULTS Diatom culture conditions: the Thalassiosira fluviatilis diatom species was cultured in a culture in accordance with the procedures described in U.S. Patent No. 5,635,493, incorporated herein by reference.

SEM Procedures: The techniques used in this study were in accordance with that described above. Purification procedures: First p-GlcNAc was purified by treatment by HF, specifically, in a fume hood, 2.42 ml of a solution of 49% HF (29N) was added to the diatom contents of the culture, at room temperature to each 1000 ml of the original cell culture volume, which resulted in a 0.07 M HF solution. The mixture was then stirred for approximately 30 seconds, causing the appearance of a persistent foam on the liquid. The content of the container will rest undisturbed for 5-6 hours to allow settlement of the heavy particles. At the end of this period of time, a layer of foam had formed while the liquid itself was divided into two layers: First, a thin layer of very dark green that was in the bottom of the container under a second layer of a gray-green color, much lighter and turbid, representing perhaps 85-90% of the total volume of the liquid. The foam layer was carefully removed through a siphon, using a capillary glass tube and vacuum suction. E. The cloudy grayish supernatant was then removed through the siphon, taking care not to disturb the dark background layer which consisted mainly of settled cell bodies, and was transferred to a separate plastic container. The cloudy grayish supernatant was allowed to stand undisturbed for an additional 16 hours. The fluid was initially almost colorless, light gray, but not transparent. After 16 hours of settling time, a small amount of foam remained on top of the main part of the liquid and a small amount of green matter had settled to the bottom of the container. The liquid was lighter in color but not yet transparent. The foam in the upper part of the liquid was removed by siphon, as before. The main room of the liquid was then carefully removed with a siphon, leaving behind the small amount of gray material settled in the bottom of the container. The liquid isolated in this way contained most of the p-GlcNAc fibers and some impurities. To remove proteins and other unwanted matter released by diatoms during the preceding steps in the process from the liquid containing fibers, the suspension of fibers and cell debris was washed with sodium dodecyl sulfate (SDS). Specifically, the required volume of a 20% SDS solution was added to form the final SDS concentration at 0.5% liquid by volume. The container containing the liquid was sealed, held in the horizontal position of a stirring machine, and stirred for 24 hours at the rate of approximately 100 agitations per minute. Shortly after the onset of agitation, large piles of white p-GlcNAc fibers appeared in the suspension in large piles, and a considerable amount of foams accumulated in the upper space of the vessels. Upon completion of the SDS wash, the contents of the containers were transferred to a Buchner filtration equipment equipped with a sulfon polyester filter membrane Supor-800 with a pore size of 0.8 μ (Gelman, Inc.). The liquid was filtered by suction, and the p-GlcNAc fibers in the liquid were collected on the filter membrane. The p-GlcNAc fibers collected in the filter membrane were then washed further. First the fibers were washed with deionized, distilled H2O, hot (70 ° C), using 3 times the volume of the original suspension, with a water jet using deionized, distilled H20, the clumps of white fibers collected in the Filter membrane of the Buchner filter were transferred to a Waring blender, and fiber blankets were disintegrated with approximately 10 short mixing pulses. The suspension of the disintegrated fibers was transferred to a Buchner filter funnel with a polyethersulfone filter membrane in accordance with that described above, and the liquid was removed with suction. The fibers collected !; were washed with 1000 ml of a hot solution (70 ° C) of IN HCl and subsequently washed further with 1000 ml of warm distilled deionized H20 (70 ° C). Finally, the fibers were washed with 1000 ml of 95% ethanol at room temperature, and filtered to dryness. The filter membrane and the filter membrane supporting the filter membrane were then dried in a drying oven at a temperature of 58 ° C for 20 minutes. The membrane and the membrane support were then placed in a drying device for 16 hours. The membrane was then carefully detached from the filter membrane. Second, p-GlcNAc was purified by using the acid / neutralization treatment method described in section 5.1., Svpra. Specifically, the p-GlcNAc was processed in accordance with that described above in this section, until before the washing step with SDS, at which point the solution was neutralized to a pH of about 7.0 by the addition of a 2.9M Tris solution. The yield of p-GlcNAc from this particular purification procedure was 20.20 milligrams per liter of diatom culture, even though, on average, approximately 60 milligrams are obtained per liter of diatom culture. A SEM micrograph of a membrane formed as a result of the purification procedure by acid / neutralization treatment appears in Figure 3. 8. EXAMPLE: DEACTIVATION of p-GlcNAc A p-GlcNAc membrane was suspended in an aqueous solution of 50% NaOH. The suspension was heated at 80 ° C for two hours. The resulting deacetylated membrane was dried and studied by scanning electron microscopy as shown in figure 5. 9. EXAMPLE: REFORMULATION of p-GlcNAc A p-GlcNAc membrane (16.2 mg) was dissolved in 1 ml of a solution of dimethylacetamine. containing 5% LiCl. The solution containing p-GlcNAc was placed in a syringe and extracted in 50 ml of pure water to precipitate the fibers. The resulting fiber material was studied using scanning electron microscopy, as shown in Figure 6. 10. EXAMPLE: PREPARATION OF A COMPOSITION OF EA / p-GlcNAc OF THE INVENTION Ro61-06i: ./ 001 (also known as "Ro61") is an endothelin antagonist not based on peptide, not specific with the structure presented in formula I, supra. Its chemical name, in the form of salt, is the sodium salt of [6- (2-hydroxy-ethoxy) -5- (2-methoxy-phenoxy), -2- [2- (lH-tetrazol-5-yl) - 5-isopropy1-pyridine-2-sulfonic acid pyridine-4-yl] -pyrimidine-4-yl] amide (1: 2) and its molecular weight is 649.59. Its solubility in water is greater than 3%. The binding inhibition potency of Ro61 (IC50) for the ETA receptor is l-20nM and as for the ETB receptor it is 2-30 nM.

Its functional inhibitory potency (pA2) in terms of the ETA receptor is 9.5 and in terms of the ETB receptor it is 7.7. Its recommended dose in vivo is 1-30 mg / kg iv or ip. Its recommended dose in vitro 10 ~ 9 to 10 ~ 5 M. Ro61 for use in a composition of the invention was initially provided in the form of a lyophilized powder by Acetelion Ltd, Allschwil, Switzerland, Switzerland, which was suspended in sterile water, and the pH was adjusted to 4.0 with sterile hydrochloric acid. Alternatively, Ro61 can be synthesized using known techniques. A p-GlcNAc fiber slurry was prepared in the following manner: p-GlcNAc prepared by the biological / chemical method described in the example section 7 above was suspended in distilled-deionized water and agitated to form a fibrous suspension or a paste of approximately 1 mg / ml. The fibrous pulp was then oven dried at a temperature of 60 ° C for two hours to form p-GlcNAc polymer membranes. The membranes were deacetylated in a 40% NaOH solution at a temperature of 80 ° C for 2 hours. When the membranes reached a deacetylation of 100%, they were washed with distilled-deionized water until achieving a pH of 7.0. The washed deacetylated membranes were then converted to p-GlcNAc lactate salt in the presence of lactic acid essentially in accordance with that described in dissolution of the p-GlcNAc latate membranes in distilled-deionized water to the desired concentration, for example, 2% p-GlcNAc-lactate, and adding R06I to the solution. The final concentration of R06I in the gel was adjusted in such a way that each animal will receive 3 mg / kg in a 200 μl gel sample. Optionally, a reactive grade propylene (2-propanediol) can be added to the p-GlcNAc solution to a final concentration of propylene glycol comprised between 1 and 10%. In some cases, a preservative can be added to prevent bacterial and / or fungal contamination. In accordance with other embodiments, concentrations of p-GlcNAc lactate within a range of 0.1% to 4.0% can be prepared in accordance with that described above. The viscosity of these preparations increases as the percentage of p-GlcNAc lactate rises such that formulations having 0.5% or more of p-GlcNAc lactate behave like gels. HE HAS. EXAMPLE: B16 AS A MODEL OF TUMOR RESPONDS TO ENDOTHELIN B16 cells were evaluated for use as a tumor model system that responds to endothelin. B16 cells, i.e., from murine B16 melanoma cell line (of fibroplasic origin) were obtained from the American Type Culture Collection (Rokville, MD) in the form of a frozen material. The cells were cultured in medium Complete (CM): RPMI 1640 (Irvine Scientific, Santa Ana CA) supplemented with 10% thermally inactivated fetal bovine serum (Summit Biotechnologies, Ft, Collins CO), penicillin (50 units / ml, streptomycin (50 μg / ml), 2 mM L-glutamine, 0.1 mM non-essential amino acids MEM (Gibco BRL, Gaithesburg MD), 1 mM sodium pyruvate, and 0.05 mM 2-mercapto tanol (Sigma Im unochemicals, St. Louis MO). a temperature of 37 ° C in a humidified 5% CO2 incubator and adjusted to 1 x 10 cells / nl every third day B16 cells were analyzed for endothelin levels and endothelin receptor (ETR) expression in the following manner: ET1 in culture supernatants of B16 was measured by competitive radioimmunoassay (RPA 545, Amersham, Milford Ma) with radioactive ligand and an antibody specific for ET1.T.E. bound and free to react with a second phase system of antibody followed by separation m A standard curve was determined by calculating the percentage of ligand bound / standard 0 (B / Bo) and the concentration of ET could be read from this standard curve. The recovery from the extraction procedure was 75 + -5% based on standards that are washed in plasma (4-20 fmol / ml). The variation between trials was 10% and the intraassay variation was 9 for the ET radioimmunoassay procedure.

Using this assay, in one experiment, it was found that baseline ET1 levels in culture supernatants of B16 d.e 24 hours were 1269 fmol / ml. The addition of lOμm cultures of either an ETA or ETB agonist or both induced an increased proliferation of B16 cells in 137%, 117%, and 164% of untreated controls, respectively, with corresponding ET1 levels of 34.01, 1158, and 34.01 fmol / ml respectively. In a subsequent experiment, the baseline levels of ET1 in 24-hour B16 culture were 597 + -58 fmol / l and, the addition of 10 μm of a non-selective agonist (ETl), a selective ETB receptor agonist ( B 3020) or both increased the proliferation of B16 cells compared to untreated controls in 154%, 116% and 141%, respectively (see Table 1, below). These data indicate that murine B16 melanoma cells express ETRs, which respond to established endothelin agonists. Table 1 Ligand (receptor)% proliferation compared to untreated controls Agonists ET1 (ETA> ETB) 154 (± 20.9) BQ 3020 (ETB) 116 (± 0.8) ET1 + BQ 3020 141 (± 4.1) In addition, B16 cells were determined to express endothelin receptors through immunofluorescence staining using the Pharmingen intracellular flow cytometry pool of antibodies with anti-cytoplasmic endothelin receptors, ie, focused towards the cytoplasmic region of the receptor (Research Diagnostics Inc., Flanders NJ) and goat anti-mouse IgG isotypic antibody controls (Sigma Immunchemicals, St. Louis MO). In accordance with this procedure, B16 cells were washed in Cytofix / Cytoperm buffer (from Pharmingen kit) and incubated for 20 minutes with a permeabilization solution (0.1% Triton X100 in 0.1% sodium citrate). The cells were then incubated at a temperature of 4 ° C in the dark for 30 minutes with primary isotype control or anti-endothelin receptor antibodies, and then washed and incubated for an additional 30 minutes with the secondary antibody labeled with FITC (IgG anti-sheep mouse; Sigma). The cells were visualized and photographed using an Axioplan research microscope (Cari Zeiss Inc. Jena Germany) equipped with a 100-watt mercury light source and a 40x plan-neufluar nal objective.3. These immunofluorescence staining experiments showed that B16 melanoma cells expressed levels Detectors of ETA and ETB receivers. Linkage assays with ET-1125 also suggest that B16 cells express ET receptors. These tests were performed as follows: vascular aortic smooth muscle cells known as AlO cells, B16 and CHO cells were suspended at a concentration of 2 x 10d cells / ml in binding buffer (50 mM Tris / HCl - pH = 7.4 , 5 mM EDTA, and 0.5% BSA) in individual tubes. AlO was used as a positive control of ETA, while CHO cells were used as a negative control, demonstrating no marked ET1 binding, which is consistent with the absence of ETR in this cell line. An aliquot of 21 μl (3- [125I] iodotyrosyl) endothelin-1 (ET1-I125, from A ercham Life Science Ine, Arlington Heights, IL) was added to each tube at a final concentration of 10"12 M. Cells labeled were then divided into aliquots in microcentrifuge tubes and 18 μl of non-labeled ET1 were added (at a final concentration of 10"8M) or HBSS The tubes were mixed and each sample was divided into aliquots of 300 μl, placed in siliconized tubes and shaken for 2.5 hours at a temperature of 37 ° C. After incubation the tubes were centrifuged at 10,000 rpm for 6 minutes, cell pellets were resuspended by vortexing in 300 μl of binding buffer and washed twice another time in link buffer. After the final wash, the cells were resuspended in 500 μl of IN NaOH, stirred for LO minutes at a temperature of 37 ° C and placed in scintillation tubes for counting in a Pcickard Cobra Auto-gamma 5000 Series gamma counting system (Model 5002) ( Packard Instrument Co., Meridea, CT). The results of these ET1 binding assays, which demonstrate that B16 cells express ETR, appear in Table 2 below. Table 2 Linkage of ET1 on tumor cells B16 AlO B16 CHO Link of ET1 * 53885 (± 2555) 25605 (± 3801) 119 (± 91) Link of ET1? On 1135 (± 120) 1398 (± 691) 346 (± 259 ) competitor ** The results are expressed as average cpm per 10d cells (± SEM). ET1-I12D was added at a final concentration of 10"12 M. The competitor was unlabeled ET1 added at a final concentration of 10" 6 M. 11B. EXAMPLE: INHIBITION BY RODI OF THE PROLIFERATION OF MELLANOMA CELLS IN VITRO Normal splenocytes do not show visible ETR levels in accordance with that detected by an immunofluorescence as described above. Due to the ease of Isolation and culture of the splenocytes, with employees therefore as control cells to evaluate the effects of Rodl on cell proliferation. Splenocytes were harvested from C57BL / 6 (H-2b) 6-8 week old male mice (Jackson Laboratories, Bar Harbor MA) as follows: spleens were removed, placed in CM, and their cells were dispersed with a 3 cc syringe plunger. The cell suspension was then filtered through a 70 μm cell strainer and the erythrocytes were lysed with a solution of ammonium chloride J isis (prepared by mixing 9 parts 8.3 g / L ammonium chloride; 20.59 g / L Tris, pH 7.65 immediately before use). The splenocytes were then washed and resuspended in CM. Rodl was obtained in the form of a lyophilized powder from Aceite Lion Ltd, Allschwil, Switzerland, in accordance with that described above and proliferation assays were carried out to evaluate the effect of Rodl on the proliferation of B16 cells in vitro. More specifically, Ro61 in HBSS was added in present concentrations to a 96-well culture plate. B16 cells or control splenocytes in accordance with that described above were then added to the wells at a concentration of 10 5 cells per well and cultured for 72 hours. Cell proliferation was assayed using the CellTiter 96 set of elements for measurement of non-radioactive cell proliferation (Promega, Madison Wl) as follows: in brief cells were incubated with 15 ml of MTT dye (from the Promega element set) for 4 hours after which the crystal formation of Formazan was visible. The crystals were dissolved in 50 ml of a solubilization / stopping solution (from the Promega element set) for 30 minutes at room temperature and color changes were measured as OD 570 nm. Background OD at 630 was automatically subtracted. The mean values of wells were determined in triplicate. The proliferation of cells or cell death was also recorded by light microscopy photography at a focal power of 40X. The inhibitory effect of Ro61 on melanoma cells 316 is illustrated in Figure 7. The proliferation of cells treated with Rod1 is expressed as a percentage of untreated control cells. The average values of wells in triplicate. As can be seen in Figure 7, Rodl inhibited the proliferation of B16 cells (filled circles) but did not inhibit normal splenocytes (empty circles). In addition, when Rodl was added in increasing concentrations to the cells in culture, a dose-dependent inhibition was observed. This effect was observed in concentrations of 0.1 uM (inhibition of 22% compared to controls not treated) and this effect was maximum at 10 μM (83% inhibition). In microscopic terms, at the highest concentration of Rodl, B16 cells no longer had a normal fibroblast-like spindle shape but were round and scanty with limited viability (see Figures 17A-17B). In contrast to this situation, the splenocytes were only minimally affected by the addition of even the highest concentration of Ro61 (10 μM). It was also found that the levels of endothelin in the culture supernatants of B16 were increased (from 513 ± 27 fmol / 1 to 50 nM of Rodl to 954 ± 31 fmol / 1 to 5 μM of Rodl) in a dose-dependent manner ( p <0.001). This observation is consistent with Rodl blocking of ETR resulting in the interruption of a receptor-mediated feedback loop. Since Rodl, an inhibitor of the ET receptor, caused the inhibition of B16 cell proliferation, it was interesting to determine if the addition of specific peptide agonists for ETA or ETB could reverse this effect, that is, by competition with Rodl for sites of receiver link. The agonist, BQ-3020- [Ac- [Alall, 15] -endothelin (6, 21), ie, BQ3020 (Novabiochem, Catalog No. Al 4534) and the agonist, [Ala1'3'11'15] -endotelinal (Sigma Irununochemicals, St. Louis MO, catalog no .: E6877) were suspended in HBSS for use. 5 x 104 B16 cells were cultured with either one of the agonists, either with both agonists together or with none of the agonists at a concentration of 10 nM in each well and Rodl was then added at various concentrations to the wells. As demonstrated in Figure 10, inhibition by Rodl of B16 proliferation was prevented by the addition of ET receptor agonists. The proliferation of cells treated with Rodl is expressed as a percentage of untreated control cells. Mean values of the wells were determined in triplicate. First, as indicated in the Y axis of Figure 10 (Rodl concentration 0), the addition of BQ3020 agonists (full triangle) or ET-1 agonist (empty rhombus) alone, or both agonists in combination (empty square), induced the proliferation of B16 cells. For the BQ3020 agonist, the proliferation was 137% of the untreated controls, for the ET1 agonist, the proliferation was 117% of the untreated controls and for the combination of the agonists, the proliferation was 164% of the controls not treated. As further indicated in Figure 10, the addition of these agonists also counteracted the inhibition induced by increasing doses of Ro61. For example, the addition of ET1 at a concentration of 10 nM completely reversed the effects of Ro61 at a concentration of 1 nM and decreased the inhibition of Rodl at 5 μM by 50%. The BQ3020 agonist could significantly reverse the effects of 5 μM Rodl (inhibition of only 12% proliferation at 5 μM). The most significant effect was observed with the combination of the two agonists inducing proliferation (123% of the untreated controls) even with the addition of 0.1 μM of Rodl. These dose-dependent findings defined a response of endothelin receptor-mediated proliferation to murine B16 cells that can be inhibited by an ETR antagonist. In other experiments, Rodl, an antagonist of both ETA and ETB which has an affinity about 10 times higher for ETA ,. was tested together with BQ123, an antagonist of ETA and BQ788, an antagonist of ETB (both obtained from American Peptide Co., Sunnyvale, CA) in order to determine the effect (ie these antagonists on the proliferation of B16 cells using the assay of cell proliferation described supra The presence of BQ123, BQ788, BQ123 + BQ788, or Ro61-0612 / 001 (each in a total concentration of 10 uM) in culture resulted in a significant inhibition of proliferation (16, 18). , 19 and 50%, respectively, compared to untreated controls, p = 0.001, see Table 3 below.) Table 3 Ligand (receptor)% proliferation compared to untreated controls Antagonists BQ 123 (ETA) 84 (± 6.6) BQ 788 (ETB) 82 (± 1.9) BQ123 + BQ 788 81 (± 4.5) R? 61 (ETA> ETB) 50 (± 4.8) In other tests of proliferation in accordance with that described above, several endothelin antagonists were tested in order to determine their effect on the proliferation of B16 cells. B16 cells that have been passed IC fold in animals and that express a full-length wild type TR, were compared to B16 cells, designated FO expressing a truncated ETA mRNA and therefore appear to produce an incomplete ETA receptor. In accordance with that illustrated in Figures 8 and 9, the endothelin antagonists, IRL, BQ123, BQ610, BQ485, BQ788, REISE, at a concentration of 10 ~ d M all inhibited the proliferation of B16 cells compared to controls FO and the endothelin agonist controls ET1 and BQ3020. 12. EXAMPLE: INDUCTION BY RODI OF THE DEATH OF APOPTOTIC CELLS OF MELANOMA TUMOR B16 In vitro studies indicated that treatment with Ro61 not only; It contributed to an inhibition of cell proliferation but was also a significant component of cell death or cell necrosis present. In fact, B16 cells treated with Rodl showed a significant degree of morphological changes consistent with programmed cell death (see Figure 11). Therefore, the effect of Rodl on the death of apoptotic cells B16 was investigated. B16 cells were grown in CM in 25 ml culture flasks with or without 1 μM of Ro61, at a temperature of 37 ° C in a 5% C02 incubator for up to 72 hours. The cells were assayed for apoptosis or cell death using the Fluorescein In Situ Cell Death Detection Kit (Boehringer Mannheim, Mannheim, Germany) as follows: in summary, cells were trypsinized and washed with PBS containing 1% bovine serum albumin (BSA). After fixation with a solution of 4% paraformaldehyde in PBS (pH 7.4) for 30 minutes, the cells were permeabilized with a 0.1% Triton X100 solution in 0.1% sodium citrate for 2 minutes on ice. The cells were then washed and labeled with a TUNNEL reaction mixture (set of Boehringer elements) for 1 hour at a temperature of 37 ° C and washed. Fluorescence was analyzed on a Coulter EPICS XL flow cytometer (Coulter, Miami FL). The measurements were compared with a positive control using 100 μg / ml of Dnase I (Boehringer Mannheim, Mannheim, Germany) for 10 days. minutes at room temperature in order to induce double-stranded DNA breaks. In accordance with that shown in Figure 12, the endothelin antagonist R06I induced apoptosis in B16 melanoma cells in culture. For example, the addition of a 1 uM of Rodl to B16 cells significantly increased the percentage of cells undergoing apoptosis compared to untreated controls (p) 0.0007). The increase in percentage of positive cells by the TUNNEL assay described above was detectable from 24 hours and was still detectable at 72 hours. The effect of duration after culture with Rodl was not significant; however, the increased apoptosis of cells treated with Rodl in comparison with untreated controls was highly significant (12.7%, 95% confidence interval: 11.7% -13.8%). These results established the presence of apoptotic cell signaling mediated through ETR that may be induced by an ETR antagonist. Thus, apoptosis is contributing at least partially to the inhibition of cell proliferation and cell death observed. 13. EXAMPLE: INHIBITION OF INTRAPERITONEAL CARCINOMATOSIS OF MELANOMA B16 IN VIVO BY AN ANTAGONIST OF ENDOTHELIN OR A COMPOSITION OF EA / p-GlcNAc OF THE INVENTION The striking effects of Rodl on B16 cells in vitro led to further studies to evaluate the impact of ETR antagonism on tumor growth in vivo using an aggressive intraperitoneal (IP) model of Bld melanoma metastasis / carcinomatosis. In accordance with this model, female C57BL / 6 mice received Ltone intraperitoneal injections of 5 X 10 4 B16 cells in 100 ml of HBSS. One day later, the mice received a 100 μl injection of either HBSS alone (no treatment), HBSS containing 3 mg / kg Ro61 (administered daily), HBSS containing 30 mg / kg Ro61 (administered daily) , p-GlcNAc gel (2%) alone, or p-GlcNAc gel (2%) containing 18 mg / kg of Rodl. Animals in the groups treated with HBSS that contained either 3 mg / kg or 30 mg / kg of Rodl alone received i.p. while all other groups were treated only once. The p-GlcNAc gel alone that was used in this experiment was prepared in accordance with that indicated in Section 10, supra, without addition of Rodl. Rodl / p-GlcNAc used in this experiment was also prepared in accordance with that presented with details above in Section 10. The mice were sacrificed after 7 days and the presence of intraperitoneal disease was evaluated. More specifically, colonies of individual tumors were counted in a dissecting microscope for each animal on the peritoneal surface, mesentery, liver, spleen and pancreas. Studies were conducted under double-blind conditions. Photographs were taken of the peritoneum and digestive organs as well as sections of the mesentery, liver, spleen and pancreatic fat and peritoneum for histological analysis by H and E as well as melanin staining. Figure 13A shows that mice that received daily PI injections of R06I at a low dose, for example, 3 mg / kg for 6 days (a total of 18 mg / kg) did not present significantly lower numbers of metastases or tumor colonies per animal. . (As used herein, the term "metastasis" refers to tumor colonies detected in the peritoneum and digestive organs of mice that received injections of B16 melanoma cells in accordance with the carcinomatosis model described above). In contrast, daily injections of higher doses of Rodl alone has tope shaking of 30 mg / kg for 6 days (a total dose of 180 mg / kq) significantly decreased the average number of tumor colonies by day 7 after tumor injection . Furthermore, in accordance with what is presented in Figure 13B, the p-GlcNAc gel only reduced the average number of tumor colonies compared to the untreated controls when it was injected IP but had no significant effect on the growth of the colonies when It was injected subcutaneously (SC). Figure 13B also shows that the greater reduction of tumor colonies was observed with the administration of a single dose (18 mg / kg) of R06I formulated in the p-GlcNAc gel (average number of colonies = 2.7 ± 1.4). The gel / rod composition resulted in a significantly lower number of colonies compared to both the high dose of R06I (p = 0.001) and compared to the gel IP administration of p-GlcNAc alone (p = 0.02). A treatment with low dose of Rodl and p-GlcNAc SC alone were not significantly different from untreated controls. To determine if Rodl did not only have a local effect due to p-GlcNAc but also a systemic effect, we investigated the effects of treatment with Rodl IP in a SC subcutaneous SC melanoma model. Female mice CS7BL / 6 received flank SC injections of 5 x 10 4 B16 melanoma cells in 100 μl of HBSS. After 24 hours, the animals received one of the following treatments (100 μl, IP): HBSS (daily x 6), 30 mg / kg of Rodl in HBSS (daily x 6), p-GlcNAc gel (one administration) or 13 mg / kg of Rodl in p-GlcNAc gel (one administration). Animals were monitored for tumor appearance and growth for a period of 3 weeks after tumor injection (n = 10 in all groups). As demonstrated in Figure 14, the B16 melanoma model SC showed that all animals that received R06I IP in HBSS developed tumors by day 17; however, a significant delay was observed in the appearance of tumors (50% of mice with tumors by day 15 compared to 100% mice with tumors by day 15 in the untreated controls). The combination of Rodl plus p-GlcNAc resulted in a delay in the appearance of tumors (50% of mice with tumors by day 13) as well as 10% of animals without tumors by day 21 after tumor injection, which suggests that the sustained release of Rodl, in a separate compartment of the tumor can significantly affect the growth of B16 melanoma. No differences in appearance and tumor growth were observed between the untreated control group and the group that received p-GlcNAc IP. Using the model of carcinomatosis described above, the effect of several other endothelin antagonists on the formation of B16 tumor colonies was evaluated. In these experiments, the animals were injected with 100 μl of samples containing 14.3 mg / ml of each drug or mixture of drugs in a single dose. As shown in Figure 15, a mixture of BQ123 (an ETA antagonist) and BQ788 (an ETB antagonist) in a final total concentration equal to that of R06I (an ETA and ETB antagonist) significantly decreased the number of colonies tumor to a degree similar to that shown by Rodl, compared to untreated control B16 cells. In addition, when the mixture of BQ was combined with p-GlcNAc in accordance with that described above, the number of tumor colonies was further decreased and to a degree almost equivalent to that observed with R06I plus p-GlcNAc. Finally, the mixed ETA / ETB antagonist, GRGDS, a commercially available pentapeptide (Americar Peptide, Co.), When combined with p-GlcNAc, demonstrated an even greater decrease in the number of tumor colonies: that Ro61 / p-GlcNAc ( see Figure 15, last column). 14. EXAMPLE: LONG-TERM SURVIVAL OF MICE C57BL / 6 AFTER MELANOMA B16 RETOINTRAPERITONEAL WITH A ENDOTHELIN ANTAGONIST ONLY OR WITH AN EA / p-GlcNAc COMPOSITION OF THE INVENTION We also evaluated endothelin antagonist therapy in long-term survival experiments in vivo. The results appear in Figure 16. Female C57BL / 6 mice received intraperitoneal injections of 5 X 10 4 B16 cells in 100 ml of HBSS. Animals were randomly separated into four groups for any of the following treatments: (a) no treatment (filled squares !; (b) 100 μl of p-GlcNAc gel alone (crosses); (c) 100 μl of HBSS daily contained 3 mg / kg of Rodl (filled triangles), or (d) 100 μl of p-GlcNAc gel containing 3 mg / kg of Rodl (empty squares). The animals were monitored daily and sacrificed for humanitarian reasons when it was determined that they were dying. It is interesting to note that B16 melanoma is an extremely virulent tumor that results in a 0% survival rate consistently within a period of 19-20 days from the tumor injection. In accordance with what is indicated in Figure 10, the injection of p-GlcNAc alone delayed death in 5 days but did not increase the survival rate of the animals. However, the combination of p-GlcNAc and Ro61 in a low dose (3 mg / kg) also delayed the death of the animals and 33% of the animals did not present evidence of tumor by day 33 after the tumor injection. Daily injections of the same low dose of Ro61 alone did not affect the survival of the mice. 15. COMMENTS OF THE RESULTS The stages performed here show direct evidence that the inhibition of endothelins binding to their receptors may affect the normal proliferation of a murine melanoma cell line, both in vivo and in vivo. For example, the endothelin antagonist, Ro61, is an inhibitor of ETA and ETB receptors with approximately 10-fold higher affinity for ETA. This correlates well with the dose-dependent inhibition of the proliferation of melanoma cells by Rodl in our experiments, as well as the more potent effect of opposition to this inhibition that is obtained by the addition of an ETA agonist unlike an ETB agonist. In addition, numerous, other endothelin antagonists here also exhibited an inhibition of melanoma cell proliferation, including specific antagonists for both ETA and ETB, for example, BQ123, BQ485, BQ610, BQ788, RES and IRL. It is interesting to note that melanoma cells express high levels of ET receptors and are more susceptible to inhibition of endothelin antagonists than normal splenocytes, which are known to have much lower membrane ET receptors. Thus, the evidence presented here indicates that endothelin antagonists such as Ro61, BQ123, BQ485, BQ610, BQ788, RES and IRL have an anti-proliferative effect on tumor cells in culture, said growth inhibition of tumor cells is mediated by binding to endothelin receptors that respond to peptide agonists specific for ETA and / or ETB. In addition, our studies indicate that endothelin antagonists alone or in combination with the p-GlcNAc described here significantly reduce carcinomatosis in vivo. In addition, the EA / p-GlcNAc compositions of this invention, for example, Rodl / p-GlcNAc, increase dramatically the survival rate of animals carrying tumor cells in vivo. The effect of R06I seems to involve an apoptotic mechanism of action, which does not contradict some of the known mechanisms of action of endothelins and their mechanisms of signal transduction. Pharmacokinetic studies with Rodl indicate that it is metabolized relatively rapidly in vivo, for example, within c.e 2-4 hours; further, when Rodl is combined with the p-GlcNAc polymer of this invention, Rodl is retained in the gel and released more slowly, and can be detected, for example, for at least 48 hours. In addition, if the concentration of the endothelin antagonist in the gel is increased, the antagonist can be retained in the gel for at least 72 hours (data not shown). Accordingly, by modifying the concentration of endothelin antagonist in the p-GlcNAc of the invention, a desired slow release can be obtained, and consequently an increased effect of the antagonist in the treatment of a proliferative disease. Finally, as indicated in Table 4 below, other types of cells expressing the ETA receptor also react with the endothelin antagonist Rodl in accordance with that described herein, ie, to exhibit an inhibition of cell proliferation upon completion. an exposure to antagon.Last of endothelin. These data are correlated, in different types of cells, with the presence for example of the ETA receptor with the effect of endothelin antagonists on cell proliferation. For example, these results indicate that if a tumor such as a pancreatic, breast or prostate tumor expresses the ETA receptor, it can be treated with an ETA-sensitive endothelin antagonist in accordance with what is disclosed herein. Table 4 ETA Rodl effect Negative control CHO No No (5/5) Melanocyte melanocyte Yes Yes (2/2) Mel noma B16F10 Yes Yes (5/5) MEL 501 No No MEL 888 Yes Yes (2/3) Pancreatic cancer ASPC 1 Yes Yes (5/5) ASPC 4 No No (5/5) CASPAN 1 No No (4/5) PANC 1 Yes Yes (4/5) BXPC 3 Yes Yes (3/4) Breast cancer MDA 231 No No (3/3) MDA 453 Yes Yes (3/3) Prostate Cancer DUI 45 No No (5/5) LN CAP No No (5/5) The present invention is not limited in its scope to the specific embodiments described herein that have only the purpose of illustrating individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are within the scope of the appended claims.

Claims (34)

1. CLAIMS An antitumor composition comprising at least one endothelin antagonist in combination with a poly-β-l- > 4-N-acetylglucosamine, said poly-β-l- > 4-N-acetylglucosamine comprises from about 4,000 to about 150,000 monosaccharides of N-acetylglucosamine covalently linked in a β-l- > 4, without protein, substantially free of other organic or inorganic contaminants, and having a molecular weight of about 800,000 da.-tones to about 30 million daltons.
2. The composition according to claim 1, wherein the endothelin antagonist is a non-specific endothelin amagonist.
3. The composition according to claim 1, wherein the endothelin antagonist is a specific endothelin antagonist for ETA.
4. The composition according to claim 1, wherein the endothelin antagonist is an endothelin antagonist specific for ETB.
5. The composition according to claim 1, wherein the endothelin antagonist is a peptide-based endothelin antagonist.
6. The composition according to claim 1, wherein the endothelin antagonist is a endothelin antagonist not based on peptides.
7. The composition according to claim 6, wherein the endothelial antagonist not based on "" -peptides is a pyrimidylsulfonamide compound.
8. 8. The composition according to claim 7, wherein the pyrimidylsulfonamide compound is Roßl.
9. 9. The composition according to claim 1, wherein the poly-β-l- > 4-N-acetylglucosamine comprises a derivative of poly-β-l- > 4-N-acetylglucosamine where At least one monosaccharide of N-acetylglucosamine has been deacetylated.
10. The composition according to claim 9, wherein the deacetylated monosaccharide is derivatized in a lactate salt.
11. 11. The composition according to claim 9, wherein from at least about 25% to about 75% of the monosaccharides of N-acetylglucosamine have been deacetylated.
12. 12. The composition according to claim 1, 0 9 or 10, wherein the poly-β-l- > 4-N-acetylglucosamine is ---____ formulated as a gel.
13. 13. I read the composition according to claim 1, or according to claim 9, wherein the poly-β-l- > 4-N-acetylglucosamine is a mat, a chain, a string, a membrane, a fiber or a sponge.
14. The composition according to claim 12, wherein the endothelin antagonist is dissolved in the poly-β-l-> gel. 4-N-acetylglucosamine.
15. 15. An antitumor composition comprising at least one endothelin antagonist in combination with a poly-β-l- > 4-N-acetylglucosamine, said poly-β-l- > 4-N-acetylglucose ina comprises from about 4,000 to about 150,000 glucosamine monosaccharides covalently linked in a β-l- > 4, without protein, substantially free of other orcanic or inorganic contaminants, and having a molecular weight of about 640,000 daltons to about 24 million daltons.
16. 16. The composition according to claim 15, wherein the endothelin antagonist is a non-specific endothelin agonist.
17. The composition according to claim 15, wherein the endothelin antagonist is a specific endothelin antagonist for ETA.
18. The composition according to claim 15, wherein the endothelin antagonist is an endothelin antagonist specific for ETB.
19. 19. The composition according to claim 15, wherein the endothelin antagonist is a Endothelin antagonist based on peptides.
20. The composition according to claim 15, wherein the endothelin antagonist is an endothelin antagonist not based on peptides.
21. The composition according to claim 20, wherein the non-peptide-based endothelin antagonist is a pyrimidylsulfonamide compound.
22. 22. The composition according to claim 21, wherein the pyrimidylsulfonamide compound is Rhod.
23. 23. The composition according to claim 15, wherein the poly-ß-l- >; 4-N-glucosamine is derived to a lactate salt.
24. 24. The composition according to claim 15 or 23, wherein the poly-β-l- > 4-N-glucosamine is formulated as a gel.
25. 25. The composition according to claim 15, wherein the poly-β-l- > 4-N-glucosamine is a mat, a string, a string, a membrane, a fiber or a sponge.
26. 26. The composition according to claim 24, wherein the endothelin antagonist is dissolved in the poly-β-l-> gel. 4-N-glucosamine.
27. 27. The composition according to claim 26, wherein the endothelin antagonist is Rodl and that of poly-β-l- > 4-N-glucosamine is a 2% gel.
28. 28. A method for the treatment of prcliferative diseases comprising administration to a of a therapeutically effective amount of at least one endothelin antagonist in 5 combination with a poly-β-l- > 4-N-acetylglucosamine, "Said poly-ß-l- > 4-N-acetylglucosamine comprises 4,000 to about about 150,000 N-acetylglucosamine monosaccharides covalently attached in a beta-conformation l- > 4, no protein, 10 substantially free of other organic or inorganic contaminants, and having a molecular weight of about 800,000 daltons to about 30 million daltons.
29. 29. A method for treating proliferative diseases comprising 15 administering to a patient a therapeutically effective amount of at least one endothelin antagonist in combination with a poly-ß-l- > 4-N-glucosamine, said poly-β-l- > 4-N-glucosamine comprises approximately 20 4,000 to about 150,000 monosaccharides from ~ "glucosamine covalently bound in a β-l-> 4 conformation, without protein, substantially free of other organic or inorganic contaminants, and having a molecular weight of about 640,000 25 tons to approximately 24 million daltons.
30. 30. The method according to claim 28 or 29, wherein the proliferative disease is cancer.
31. 31. A pharmaceutical composition for the treatment of a proliferative disease, comprising an amount Therapeutically effective T of a non-peptide-based endothelin antagonist in a pharmaceutically acceptable carrier
32. 32. The composition according to claim 31, wherein the endothelin antagonist is a 10 ~ pyrimidylsulfonamide compound.
33. 33. The composition according to claim 31, wherein the proliferative disease is cancer.
34. 34. A method for the treatment of cancer, comprising administering to a patient an amount Therapeutically effective of a non-peptide-based endothelin antagonist in combination with a pharmaceutically acceptable carrier. __ - 35 The method according to claim 34, wherein the endothelin antagonist is a compound 20 of pyrimidylsulfonamide. 25