MXPA99009543A - Alkaloid halide salts of swainsonine and methods of use - Google Patents

Abstract

Crystalline salts of swainsonine, and methods using same.

Description

, £. HALURO SALTS OF ALCALOID OF SWAINSONINA AND METHODS OF USE FIELD OF THE INVENTION The invention relates to swainsonine halide salts and methods of using the salts.

BACKGROUND OF THE INVENTION Swainsonin (SW) is an indolizidine alkaloid which can be isolated from Australian Swainsona canescens (Colegate et al., Aust J. Chem 32: 2257-2264, 1979), North American plants of the genus Astragalua and Oxytropis (Molyneux RJ and James LF., Science 215: 190-191, 1981), and the fungus Rhizoctonia leguminicola (Schneider et al., Tetrahedron 39: 29-31, 1983). Swainsonin has interesting immunomodulatory and cancer suppressive activity which has been attributed to its ability to inhibit a-mannosidase II activity. It is considered that swainsonin works as an enzyme inhibitor because it can mimic the The glycosylate cation intermediate is generated during the hydrolytic cleavage of mannopyranosides (Goss, P.E. et al., Clin Cancer Res. 1: 935-944, 1995). Swainsonine blockade of arsenidase II prevents the expression of bound carbohydrates N Branched GlcNAc ß (1-6). It has been found that murine tumor cells treated with swainsonin are less metastatic both in organ colonization and in spontaneous metastasis assays in mice (Dennis JW, Cancer Res. 46: 5131-5136, 1986 and Humphries et al., Proc. Nati, Acad. Sci. USA 83: 1752-1756, 1986). It has also been shown that swainsonin blocks the invasion of tumor cells through the extracellular matrix in vitro (Yegel et al., Int. J. Cancer 44: 685-690, 1989 and Seftor et al., Melanoma Res. 1: 53-54, 1991). Swainsonin administered orally or by miniosmotic bombs to athymic nude mice inhibited the growth rate of human MeWo melanoma and HT29m colon carcinoma tumor stenoses in mice (Dennis et al., J. Nati. Cancer Inst 81: 1028 -1033, 1898 and Dennis et al., Cancer Res., 50: 1867-1872, 1990). In Canada, phase 1 clinical trials of swainsonin have been completed in patients with metastatic cancer (Goss et al., Cancer Res., 54: 1450, 1995 and Goss et al., Clinical Cancer Research, 3: 1077, 1997). Swainsonin has immune stimulatory effects (reviewed in Humphries M.J. and Olden K., Pharmacol Ther 44: 85-105, 1989 and Olden et al., Pharmacol Ther 50: 285-290, 1991)). In particular, swainsonine has been shown to alleviate immune suppression both chemically induced and associated with tumor (Hiño et al., J. Antibiot (tokyo) 38: 926-935, 1985), the increase in NK cells (Humphries et al. ., Cancer Res. 48: 1410-1415, 1988), and activities of LAK cells (Yagita M. and Saksela E., Scand J. Immunol., 31: 275-282, 1990), and increase the proliferation of splenic cells and of bone marrow (BM) (White et al., Biochem. Biophys., Res. Commun. 150: 615-625, 1988; Bowlin et al. Cancer Res 49, 4109-4113, 1989, and White et al., Cancer Commun 3: 83-91, 1991). It has also been shown that swainsonin has hemo-restorative / chemoprotective effects. For example, swainsonine has been shown to protect against the lethality of various chemotherapeutic agents (Oredipe et al., 1991, Nati. Cancer Inst. 83: 1149-1156, 1991). In these studies, survival in mice treated with swainsonin correlates with stimulation of bone marrow proliferation, bone marrow cellularity and grafting efficiency in mice (Oredipe et al., 1991; White et al, 1991). U.S. Patent No. 4,857,315 discloses compositions containing SW and active analogues of SW in a pharmaceutical formulation to inhibit cancer metastasis and cell proliferation, and in combination with interferon or an interferon inducer, to improve the antiproliferative and antiviral effects of interferon or the interferon inducer.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to stable and substantially purified synthetic halide salts of swainsonin. Halide salts can be very difficult to purify in a stable way, and there is uncertainty that the salts of swainsonin form crystals that can be used to determine their structure by X-ray diffraction. In particular, the present inventors were able to obtain stable and substantially purified crystalline chloride and bromide salts of brominin and for determine its structure by X-ray crystallography. The swainsonine salts of the invention have anticancer activity in vitro and in vivo. Significantly, certain salts of the invention have improved stability properties compared to the free base of swainsonin, and have properties which allow them to dissolve and target their target more rapidly than swainsonin. Therefore, the salts of the present invention provide improved pharmaceutical compositions. An aspect of the invention resides in obtaining certain salts of swainsonine halide, and in particular in obtaining crystalline bromide and bromide salts of swainsonine of sufficient quality to determine the three-dimensional (tertiary) structure of the compounds by X-ray diffraction methods. Accordingly, the invention provides crystals of sufficient quality to obtain a determination of the three-dimensional structure of the salts of chloride and bromide of swainsonin with high resolution. Therefore, the present invention provides stable crystalline chloride and bromide salts of swainsonin. In particular, the invention relates to a stable chloride or bromide salt of swainsonin which comprises molecules of chloride salts or swainsonine bromide in the unit cell which are held together by hydrogen bonding interactions. In one embodiment, the chloride and crystalline bromide salt comprises four molecules of chloride salts or swainsonine bromide in a unit cell. Preferably, the chloride and crystalline bromide salt comprises molecules of the hydrochloride or hydrobromide salts of swainsonin. The salts of chloride and bromide of swainsonin of the invention, in particular the salts of crystalline hydrochloride or hydrobromide of swainsonine, can be used to prepare pharmaceutical compositions. Therefore, the invention provides a method for preparing a pharmaceutical composition comprising mixing a swainsonin chloride or bromide salt, preferably a hydrochloride salt or crystalline swainsonin hydrobromide, in a selected carrier, excipient or pharmaceutical diluent, and optionally adding other therapeutic agents.

The invention also contemplates a composition, in particular a pharmaceutical composition, comprising the chloride salt or swainsonin bromide of the invention, preferably a hydrochloride or hydrobromide salt. In a preferred embodiment of the invention, a solid is provided from the pharmaceutical composition (e.g. tablets, capsules, powdered or powdered form) comprising a hydrochloride salt or crystalline swainsonine hydrobromide. In vitro and in vivo studies have shown that the salts of the present invention, in particular the swainsonine hydrochloride salt of the invention, have immunomodulatory and cancer suppressing properties and hemo-restorative / chemoprotective properties. For example, treatment with a swainsonine hydrochloride salt of the invention reduces the growth of mammary adenocarcinoma cells SPl.A3a injected into immune competent mice, when administered either by i.p. or orally in water to drink. The growth of SPlA3a cells in vitro is stimulated by TGF-β1 and TNFa, and these effects are suppressed by the swainsonine hydrochloride salt of the invention. In addition, the treatment of murine bone marrow cells in vitro with the swainsonine hydrochloride salt of the invention stimulates the proliferation of both erythroid and granulocyte-macrophage colony forming units (CFU-E and CFU-GM, respectively).

Therefore, the invention is further related to a method for stimulating the immune system, stimulating the growth of hematopoietic progenitor cells, treating proliferative disorders of microbial or parasitic infections, or conferring protection against chemotherapy and radiation therapy in a subject. , which comprises administering an effective amount of the swainsonine salt of the invention. The invention also relates to the use of the swainsonine salt of the invention in the preparation of a medicament for stimulating the immune system, stimulating the growth of hematopoietic progenitor cells, or conferring protection against chemotherapy and radiation therapy in a subject and / or to treat proliferative disorders, and microbial or parasitic infections. The knowledge obtained regarding the salts of chloride and bromide of swainsonin can be used to model the tertiary structure of related compounds, ie, analogues or derivatives of swainsonin and salts thereof. In addition, knowledge of the structure of the chloride and bromide salts of swainsonin provides a means to investigate the mechanism of action of these compounds in the body. For example, the ability of the compounds to inhibit a-mannosidase II activity can be predicted by various computer models. The knowledge of the atomic coordinates and the atomic details of the salts of chloride and bromide of swainsonina can be used to design, evaluate computationally, synthesize and use modulators of swainsonina and analogues and derivatives thereof, that avoid or treat any physical property and undesirable pharmacological of swainsonin. Accordingly, another aspect of the invention is to provide material which is an initial material in the reasoned drug design which mimics the action of the swainsonin halide salts. These drugs can be used as therapies that are beneficial in the treatment of immune and proliferative diseases, or of microbial or parasitic infections. These and other aspects of the present invention will become apparent upon reference to the following detailed description and the accompanying drawings. In addition, various publications are referred to herein, which are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to the drawings, in which: Figure 1 shows the molecular structure of the swainsonin hydrochloride salt; Figure 2 shows the molecular structure of the swainsonin hydrobromide salt; Figure 3 is a crystal packing diagram for swainsonin hydrochloride; Figure 4 is a crystal packing diagram for swainsonin hydrochloride Figure 5 is a mass spectrum of the swainsonin hydrochloride salt of the invention; Figure 6 is a high resolution liquid chromatogram of a swainsonine hydrochloride salt of the invention; Figure 7 is a graph showing the effect of swainsonine hydrochloride on the proliferation of SP1.A3, a proliferation of mammary tumor cell in vitro; Figure 8 is a graph showing the inhibition of tumor growth by swainsonin hydrochloride by means of an Alzet pump; Figure 9 is a graph showing the inhibition of tumor growth by oral administration of swainsonin hydrochloride; Figures 10A, 10B and 10C are spots showing that swainsonin hydrochloride increases the activation of STATl in vessel after treatment of DBA / 2 mice with poly IC; and Figure 11 is a graph showing the in vitro effect of swainsonine hydrochloride on murine bone marrow CFU-GM in the presence of different cytokines.

DETAILED DESCRIPTION OF THE INVENTION Swainsonine Salts of the Invention The present invention provides stable and substantially purified halide salts of swainsonin. A "halide salt" is a salt of chloride, fluoride, bromide and iodide, preferably a chloride or bromide salt. The countercation of the salt can be an alkali metal (for example Li, Na or K), or preferably hydrogen. In one embodiment of the invention, a swainsonin hydrochloride salt having greater thermal stability than the free base of swainsonin is provided (eg, it is more stable than the free base of swainsonin when exposed to atmospheric oxygen or nitrogen at about 105. ° C for approximately seven days). In another embodiment, the present invention provides a crystalline chloride or bromide salt of swainsonin. A chloride or bromide salt of swainsonin may comprise molecules of chloride salts or swainsonine bromide in a unit cell held together by interactions of hydrogen bonds. In particular, the crystalline chloride or bromide salt comprises four molecules of swainsonine chloride or bromide salts in a unit cell. Preferably, the crystalline chloride or bromide salt comprises four molecules of the hydrochloride or hydrobromide salts of swainsonin in a unit cell. A crystalline swainsonine chloride salt of the invention can be held together by hydrogen bonding interactions from a protonated nitrogen and hydroxyl oxygen atoms of a molecule of a salt of chloride of swainsonin to chloride ions of other salt molecules of swainsonine chloride. A crystalline swainsonine bromide salt of the invention can be held together by hydrogen bonding interactions from the hydroxyl oxygen atoms of a first molecule of the swainsonine bromide salt to bromide ions of other bromide salt molecules of swainsonin, and a hydrogen-binding interaction from a protonated nitrogen atom of the first molecule of a swainsonine bromide salt to an oxygen atom of a second molecule of a swainsonine bromide salt. Preferably, a crystalline swainsonine hydrochloride salt is provided which comprises molecules of the swainsonine hydrochloride salt in a unit cell which is held together by hydrogen bonding interactions from the protonated nitrogen and the hydroxyl oxygen atoms of a salt molecule of swainsonine hydrochloride to chloride ions of other molecules of swainsonine hydrochloride salts. In addition, a crystalline swainsonine hydrochloride salt which comprises swainsonin hydrobromide salt molecules is provided in a unit cell which is held together by hydrogen bonding interactions from the hydroxyl oxygen atoms of a first molecule of the salt of swainsonine hydrobromide to bromide ions of other salt molecules of swainsonine hydrobromide, and a hydrogen-binding interaction from the protonated nitrogen atom of the first molecule to an oxygen atom of a second molecule of the swainsonine hydrobromide salt. The crystal can take any form of crystal symmetry based on the type of the halide salt molecule, the hydrogen bonding interactions and / or the spatial group. The form of symmetry is defined by the "unitary cell" which is the basic parallelepiped that is repeated in each direction to form the crystal lattice. The term "space group" refers to the arrangement of the elements of symmetry of a crystal. In one embodiment of the invention, a crystalline swainsonine hydrochloride or hydrochloride salt has a space group symmetry P212121. In a preferred embodiment of the invention, the swainsonin chloride or bromide salt comprises orthorhombic unit cells. The diffraction data obtained from X-ray crystallography is used to calculate the electronic density map of a repeated unit of the crystal. Electron density maps are used to establish the positions of individual atoms within the unit cell of the crystal. The axial lengths of the unit cell are represented by (a b e) where a = x axis, b = y axis, and c = z axis. In addition, (x and z) represent the coordinates for each atom measured as the distance along the coordinate axes a, b or c, from a point of origin. Those familiar with the art will understand that the set of atomic coordinates determined by X-ray crystallography are not found without standard error. The unit cell for a crystal of a swainsonine hydrochloride salt of the invention can have the unit cell lengths a = 8.09 + 0.01 A, b = 9.39 + 0.01 A and c = 13.62 +. 0.01 Á. The unit cell for a crystal of a swainsonine hydrobromide salt of the invention can have unit cell lengths of a = 8.40 ± 0.01 A, b = 8.63 +, 0.01 A and c = 14.12 +. 0.01 Á. In a preferred embodiment of the invention, the atoms in a crystal of a swainsonine hydrochloride salt have the atomic coordinates shown in Table 1. In another preferred embodiment of the invention, the atoms in a crystal of a hydrobromide salt of swainsonina have the atomic coordinates shown in table 2. Figures 1 and 2 show the three-dimensional structures of the hydrochloride and hydrobromide salts of swainsonina expressed using the x, y and z coordinates, respectively. The glass packing diagrams for the crystalline hydrochloride and hydrobromide salts of swainsonin are shown in Figures 3 and 4, respectively.

Preparation of swainsonine salts of the invention A crystalline salt of the invention can be prepared by treating a swainsonin acetonide with an acid and purifying the salt by crystallization. The swainsonin acetonic can be obtained as described by Bebbett et al and Cha et al (J. Am. Chem. Cos. 111: 2580-2582, 1989 and U.S. Patent No. 5,187,279, respectively). Acetone can be hydrolyzed to form a substantially pure crystalline salt of the invention. For example, a substantially pure crystalline hydrochloride salt can be formed by hydrolysis of the acetonic of swainsonin as described in Example 1. In preparing the compounds of the invention, conventional protecting groups can be used to block the reactive groups. Suitable blocking and unblocking schemes are known to those familiar with the art (see T.W. Greene and P.G.M. Wuts, 2d ed., Protective Groups in Organic Synthesis, John Wiley &Sons, New York, 1991). In general, particular protecting groups are selected which suitably protect the reactive groups in question during the subsequent synthesis steps and which are easily removable under conditions which will not cause degradation of the desired product. In vivo, some protecting groups are cut or metabolically converted to the active functional group (for example, by hydrolysis or oxidation). In some cases, metabolically cleaved protective groups are preferred. Examples of protecting groups that can be used include hydroxyl protecting groups, carboxylate protecting groups and carbonyl protecting groups. Representative hydroxyl protecting groups that can be used include the following. Methyl ethers including methoxymethyl, methylthiomethyl, t-butylthiomethyl; (phenyldimethyl ildyl) methoxymethyl; benzyloxymethyl; p-methoxybenzyloxymethyl; (4-methoxyphenoxy) methyl; guayacolmethyl; t-butoxymethyl; 4-pentynyloxymethyl; siloxymethyl; 2-methoxyethoxymethyl; 2, 2, 2-trichloroethoxymethyl; bis (2-chloro-ethoxy) methyl; 2- (trimethylsilyl) ethoxymethyl; tetrahydropyran-2-yl; 3-bromotetrahydropyran-2-yl ?; 1-methoxycyclohexyl; 4-methoxy-tetrahydropyran-2-yl; 4-methoxy-tetrahydrothiopyran-2-yl; 4-methoxytetrahydrothio-pyran-2-yl-S, S-dioxide; 1- [(2-chloro-4-methyl) phenyl] -4-methoxy-piperidin-4-yl; 1, -dioxan-2 -il; tetrahydrofuranyl; tetrahydrothiofuranyl; and 2, 3, 3a, 4, 5, 6, 7, 7a-octahydro-7,8,8-trimethyl-, 7-methanobenzofuran-2-yl. The ethylethers include 1-ethoxyethyl; 1- (2-chloroethoxy) ethyl; 1-methyl-l-methoxyethyl; 1-methyl-l-benzyloxy-2-fluoroethyl; 2, 2, 2-trichloroethyl; 2-trimethylsilylethyl; 2- (phenylenyl) ethyl; t-butyl; allyl; p-chlorophenyl; p-methoxyphenyl; and 2,4-dinitrophenyl. Benzyl ethers include benzyl; p-methoxybenzyl; 3, 4-dimethoxybenzyl; o-nitrobenzyl; p-nitrobenzyl; p-halobenzyl; 2,6-dichlorobenzyl; p-cyanobenzyl; p-phenylbenzyl; 2- and 4-picolyl; 3-methyl-2-picolyl-N-oxide; diphenylmethyl; p, p-dinitrobenzhydryl; 5-dibenzosuberil; triphenylmethyl; α-naphthyldiphenylmethyl; p-methoxyphenyldiphenylmethyl; di (p-methoxyphenyl) phenylmethyl; tri- (p-methoxyphenyl) methyl; 4- (41-bromophenazyloxy) phenyldiphenylmethyl; 4,4 ', 4"-tris (4,5-dichlorophthalimido-phenyl) methyl; 4,4', 4" -tris- (levuinoyloxyphenyl) methyl; 4,4 ', 4"-tris (benzoyloxyphenyl) methyl; 3- (imidazol-1-ylmethyl) bis (4,4', 4" -dimethoxyphenyl) -methyl; 1,1-bis (4-methoxyphenyl) -1 '-phenylmethyl; 9-anthryl; 9- (9-phenyl) xanthenyl, 9- (9-phenyl-10-oxo) anthryl; 1,3-benzodithiolan-2-yl; and S, S-bencisotazolyl dioxide. Silylesters include trimethylsilyl triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl diethylisopropyl silyl; dimethyl texillil; t-butyldimethylsilyl t-butyl-diphenylsilyl; tribenzylsilyl; tri-p-xylsilyl triphenyl silyl; diphenylmethylsilyl; and t-butylmethoxyphenylsilyl. Esters include, formate, benzoylformate; acetate; chloroacetate; trichloroacetate; methoxyacetate; triphenylmethoxyacetate; phenoxyacetate; p-chlorophenoxyacetate; p- (phosphate) phenylacetate; 3-phenylpropionate; 4-oxopentanoate (levulinate); 4, 4 - (ethylendithio) pentanoate; pivaloate; adamanthoate; crotonate; 4-methoxyrotonate; benzoate; p-phenylbenzoate and 2, 6-trimethylbenzoate. The carbonates include methyl carbonate; 9-fluorenyl-methylcarbonate; ethyl carbonate; 2, 2, 2, -trichloroethyl carbonate; 2- (trimethylsilyl) ethyl carbonate; 2 - (phenylsulfonyl) ethyl carbonate; 2- (triphenylphosphonium) -ethyl carbonate; isobutyl carbonate; vinyl carbonate; allyl carbonate; p-nitrophenyl carbonate; benzyl carbonate; p-methoxybenzyl carbonate; 3,4-dimethoxybenzyl carbonate; o-nitrobenzyl carbonate; p-nitrobenzyl carbonate; S-benzyl thiocarbonate; 4-ethoxy-1-naphthyl carbonate and methyl dithiocarbonate. Protective groups with assisted cutting include 2-iodobenzoate; 4-azidobutyrate; 4-nitro-4-methylpentanoate; 0- (dibromomethyl) enzoate; 2-formylbenzenesulfonate; 2- (methylthiomethoxy) ethyl carbonate; 4- (Methylthiomethoxy) -butyrate and benzoate 2- (methylthiomethoxymethyl). The various esters include 2,6-dichloro-4-methylphenoxyacetate; 2,6-dichloro-4- (1,1,3,3-tetramethyl-butyl) phenoxyacetate; 2,4-bis (1,1-dimethylpropyl) phenoxy acetate; chlorodiphenylacetate; isobutyrate; monosuOHCinoate; (E) -2-methyl-butenate (tigloate); o- (methoxycarbonyl) benzoate; p-benzoate; a-naphthoate; nitrate; alkyl N, N, N ', N', -tetramethylphosphorodiamidate; N-phenylcarbamate; borate; dimethylphosphinothioyl; and 2,4-dinitrophenyl-sulfenate.

Sulfonates include methanesulfonate (mesylate); benzyl sulfonate; and tosylate. Acetals and cyclic ketals include methylene; ethylidene; 1-t-butylethylidene; l-phenylethyldidene; 4- (methoxyphenyl) ethylidene; 2, 2, 2-trichloroethylidene; acetonide (isopropylidene); cyclopentylidene; cyclohexylidene; cycloheptylidene; benzylidene; p-methoxybenzylidene; 2,4-dimethoxybenzylidene; 3, 4-dimethoxybenzylidene; and 2-, 3- or 4-nitrobenzylidene. Cyclic ortho esters include methoxymethylene; ethoxymethylene, di ethoxymethylene; 1-methoxyethylidene; 1-ethoxyethylidene; 1,2-dimethoxy-ethylidene; α-methoxybenzylidene; 1- (N, N-dimethylamino) ethylidene derivative, a- (N, N-dimethylamino) benzylidene derivative; and 2-oxacyclopentylidene. These cyclic orthoesters, like the bivalent organic moieties mentioned above for adjacent pairs of substituents, can react with non-adjacent hydroxyl portions. For example, a bivalent organic moiety mentioned in the preceding paragraph or mentioned above for adjacent pairs of substituents may be selected from two non-adjacent substituents on the same molecule or for any two substituents on two separate molecules. The two separate molecules may be the same or different, and are selected from compounds described herein.

Silyl derivatives include di-t-butylsilylene groups; 1, 3 - (1, 1, 3, 3-tetraisopropyldisiloxanilidene) derivative, tetra-t-butoxydisiloxane-1,3-dilidene derivative; cyclic carbonates; cyclic boronates; ethyl boronate and phenyl boronate. Preferred protecting groups for catechols include acetals and cyclic ketals such as methylene, acetonide, cyclohexylidene and diphenylmethylene; and cyclic esters such as cyclic borate and cyclic carbonate. The invention also encompasses compounds identical to the swainsonine salts of the invention except that one or more conventional protecting groups are used, such as the hydroxyl protecting groups, carboxylate protecting groups and carbonyl protecting groups described herein. The invention further encompasses other hydroxyl protecting groups of .10 not individually identified therein which are pharmaceutically acceptable, and are optionally metabolized (eg, cut or modified) to form one of the compounds described herein. In other words, the invention encompasses metabolic precursors of the disclosed compounds and the metabolites of the disclosed compounds having anticancer, antiviral or antiproliferative activity. further, the invention encompasses quaternary amine salts and other organic salts of the described compounds, including benzenesulfonate, benzoate, citrate, lactate, tartrate, preferably formate and acetate, or other salts of carboxylic, aminocarboxylic or polycarboxylic acids. The crystals of the invention can also be formed by, for example, dissolving the hydrochloride salt or swainsonin hydrobromide in a solvent (for example methanol) and evaporating the solvent. The crystals can also be prepared by diffusion using standard methods. It will also be appreciated that the crystalline chloride or bromide salts (particularly the hydrochloride or hydrobromide salts) of functional derivatives of swainsonin can be prepared using the methods described herein, and the salts prepared by the methods contemplated by the present invention. . A "functional derivative" of swainsonin refers to a compound that possesses a biological activity (functional or structural) that is substantially similar to the biological activity of swainsonin. The term "functional derivative" is intended to include "variants", "analogues" or "chemical derivatives" of swainsonin. The term "variant" refers to a molecule substantially similar in structure and function to the swainsonin or a part thereof. A molecule is "substantially similar" to swainsonin if both molecules have substantially similar structures, or if both molecules possess similar biological activity. The term "analogue" refers to a molecule substantially similar in function to a molecule of swainsonin.

The term "chemical derivative" describes a molecule that contains additional chemical portions which are not normally part of the base molecule.

Compositions of the Invention The invention provides pharmaceutical compositions formulated from a swainsonine salt of the invention ^ (for example a chloride or bromide salt, preferably a hydrochloride or crystalline hydrobromide, more preferably an orthorhombic hydrochloride salt of swainsonin), a combination of the swainsonine salts of the invention, or a combination of swainsonin and salt or swainsonin salts of the invention. The compositions include a swainsonine salt of ^^ 15 the invention, or include a form of swainsonin prepared from a salt described, such as tablets, capsules including a soft gelatin capsule or a powdered or powdered form of the halide salt or other forms of parenteral aistration , transdermal, intranasal or oral, known in the technique. A preferred composition of the invention is a composition in solid form wherein the active ingredient, ie, the salt of the invention is in crystalline form. For example, the composition may be in the form of a tablet, capsule or powder. A composition of the invention in particularly preferred solid form having enhanced stability properties comprises a crystalline hydrochloride salt in the invention. The crystalline salts of the present invention allow the use of a substantially pure active ingredient in pharmaceutical compositions. The term "substantially pure" includes a purity of at least 95% and preferably at least 97% by weight (eg, from at least 99% to 99.5% by weight). Impurities include by-products of synthesis or degradation. A crystalline hydrochloride salt substantially of swainsonin is visually colorless and may be in the form of prisms. A composition of the invention includes one or more pharmaceutical carriers, and optionally one or more bioactive agents. For example, compositions formulated from a swainsonine salt of the invention may include: (a) a tablet including a swainsonin salt of the invention, a pharmaceutical carrier and may also include an absorption enhancer, (b) a capsule containing a crystalline, amorphous or a vitro powder, microspheres or granules made from the swainsonine salt of the invention, although in the capsule, the swainsonin salt is no longer in the form of transparent crystals (for example prism), (c) a soft gel capsule manufactured / swainsonine salt of the invention, (d) an aqueous solution of a swainsonine salt of the invention, wherein the salt of dissolved swainsonin no longer constitutes transparent crystals, and for example can no longer be associated with hydrogen or chloride or bromide, and (e) other forms of parenteral, transdermal, intranasal or oral aistration known to those familiar in the art. The free base of swainsonin derived from a salt of the invention is also useful in certain methods of treatment of the invention. The free base of pure swainsonin, however, is not contemplated for use in a composition of the invention. Routes of aistration include oral, pulmonary, topical, in a body cavity (eg, nasal, ocular, buccal), transdermal and parenteral (eg, intravenous, intramuscular and subcutaneous). The externally activated drug delivery systems include those activated by heat, ultrasound, electric pulses, iontophoresis, electrophoresis, magnetic modulation and light. Formulations include solids (tablets, soft or hard gelatine capsules), semi-solids (gels, creams) or liquids (solutions, colloids or emulsions), preferably solids. Colloidal carrier systems include microcapsules, emulsions, microspheres, multilamellar vesicles, nanocapsules, unilamellar vesicles, nanoparticles, microemulsions and low density lipoproteins. Formulation systems for parenteral administration include lipid emulsions, liposomes, mixed micellar systems, biodegradable fibers and fibrin-gels, as well as biodegradable polymers for implantation. Formulation systems for pulmonary administration include metered dose inhalers, powder inhalers, solutions for inhalation and liposomes. A composition for sustained release (multiple unit of particle disintegration of spheres, single unit of a non-disintegrating system) of controlled release (oral osmotic pump) and bioadhesives or liposomes can be formulated. Controlled release formulations include those which release intermittently and those that release continuously. Pharmaceutical carriers include inorganic substances such as calcium phosphate and titanium dioxide; carbohydrates such as lactose monohydrate and -cyclodextrin; surfactants such as sodium lauryl sulfate and poloxamers; polymers such as starch, ethylcellulose, hydrogels and polyacrylic acids; lipids such as polylactides, stearic acid, glycerides and phospholipids; or amino acids and peptides such as leucine and low density lipoprotein. The composition is formulated so that it remains active at physiological pH. The composition can be formulated in the pH range of 4 to 7. In one embodiment of the invention, a composition is provided which is an oral dosage form comprising a swainsonine salt of the invention (preferably the hydrochloride salt or crystalline hydrobromide) and a non-hygroscopic, inert and preferably anhydrous excipient (for example lactose or mannitol). In another embodiment, a composition is provided which is a soft gelatin capsule comprising a swainsonine salt of the invention (preferably a crystalline hydrochloride or hydrobromide salt) and at least one hydrophilic carrier (eg, glycerin or propylene glycol) and at least one lipophilic carrier (for example PEG 400). The compositions may also include absorption enhancers, particle coatings (e.g., enteric coatings), lubricants, steering agents and any other agent known to those familiar in the art. A composition may contain from about 0.1 to 90% by weight (eg, about 0.1 to 20% or about 0.5 to 10%) of the active ingredient. The percentage of active ingredient in each pharmaceutical composition and the effective amount of the active ingredient used to practice the present invention for the treatment of the described conditions depends on the manner of administration, the age and the body weight of the subject as well as the condition of the subject _that will be treated, and finally decided by the attending physician or veterinarian. Such amount of compound is determined by the attending physician or veterinarian is referred to herein as the "effective amount". Based on the studies of Goss et al., (1994 and 1996) with free base of swainsonin at a dose of less than 300 μg / kg / day, preferably 150 μg / kg / day or less, more preferably one dose of 75 μg / kg twice a day, or less, will be well tolerated in humans. The salts of the invention are indicated as therapeutic agents either alone or together with other therapeutic agents or other forms of treatment (eg chemotherapy or radiotherapy). For example, the compounds can be used in combination with antiproliferative agents, antimicrobial agents, immunostimulatory or anti-inflammatory agents. In particular, the compounds can be used in combination with, and can improve the activity of antiviral and / or antiproliferative agents such as the Thl cytokine. Thl cytokines include interleukins-2 and 12 (IL-2, IL-12), and interferons a, β,? (IFN-a, IFN-β, INF-?) And inducers thereof. The compounds of the invention can be used with poly (I.C.), poly (I.C.) - LC, tumor necrosis factor (TNF), or transforming growth factor (TGF). The compounds can be used in combination with chemotherapeutic agents including doxorubicin, 5-fluorouracil, cyclophosphamide and methotrexate, with isoniadiza for the prevention and treatment of peripheral neuropathy and with NSAI for the prevention and treatment of gastroduodenal ulcers. The compounds of the invention can be administered concurrently, separately or sequentially with other therapeutic agents or therapies.

The subjects to whom the composition of the invention can be administered include animals, and include mammals and particularly humans. The animals also include foster domestic animals for administration or as pets, including horses, cows, sheep, birds, fish, pigs, cats, dogs and zoo animals. The swainsonine salts of the invention can be converted into pharmaceutical compositions using standard methods. For example, a crystalline swainsonine hydrochloride or hydrochloride salt of the invention can be mixed in a selected pharmaceutically acceptable carrier, excipient or diluent, as described herein.

Inhibition of mannosidase The compounds of the invention, in particular the crystalline swainsonine hydrochloride and hydrobromide salts and the compositions made therefrom, inhibit the Golgi mannosidase II enzyme. The general inhibition of mannosidase of the compounds of the invention can be conformed by inhibition of direct measurement of Jack Bean, Golgi a-mannosidase or lysosomal (see example 18 for protocols). Mannosidase inhibition can also be tested using a L-PHA toxicity assay. The assay is based on the finding that specific binding of the toxic plant lectin L-PHA to transformed cell lines such as MDAY-D2 tumor cells is a specific measure of inhibition of oligosaccharide processing. The measurement of the IC 50 in the toxicity test of L-PHA reflects the ability of the compound to enter the cells and to carry out the inhibition of the processing of oligosaccharides. It is a general mesh for activity in cells which measure cell entry, inhibition of the target enzyme and the resulting cellular phenotype. The L-PHA assay generally involves growing transformed cells in the presence of L-PHA and the compound; measure the cell viability and / or the amount of proliferation of the cells; and determining the ability of the compound to inhibit the processing of N-linked oligosaccharides by comparing the amount of proliferation of the cells and / or cell viability with the amount of proliferation observed for the growth of the cells in the presence of L-PHA alone. Transformed cells which can be used in this assay include MDAY-D2, L1210, CHO, B16, melanoma tumor cells and human tumor cells such as SW 480, LS174T, HT-29, WiDr, T2, MDA-231, MCF7, BT-20, Hs578T, K562, Hs578T, SK.BR-3, CY 6T, MDA-468, H23, H157, H358, H1334, H1155, H28, H460; Hmesol, H187, H510A, N417, H146, H1092, H82 (Restifo, N. P. et al., J. Expert, Med. 177: 265-272, 1993). The amount of proliferation of the cells can be measured using conventional techniques. For example, cell proliferation can be measured by measuring the incorporation of labeled thymidine. More particularly, the radioactively labeled thymidine can be added for about 2-5 hours, preferably 3-4 hours and the cells can be harvested and the radioactivity can be counted using a scintillation counter. A fully automated enzymatic method based on the measurement of alkaline phosphatase activity can be used to analyze the inhibition of mannosidase II. The method is based on the observation that there is a strong correlation between the number of surviving cells and their concentration of alkaline phosphatase activity. The method uses a colorimetric assay to monitor cell proliferation of transformed cells after treatment with L-PHA. The reaction mixture is added directly to cells that grow in their own medium, so the sedimentation and washing steps of the cells are not required. Therefore, the method can be carried out in a single step, without removal of the culture medium or sedimentation and cell washing, whereby fully automated procedures are allowed. The reaction is linear with time over a broad time interval (5-180 min), and the K_ value of the enzyme for the paranitrophenyl phosphate substrate is relatively low (0.81 mM). The incubation time and substrate concentration can be changed in order to modulate the reaction rate and adjust the protocol, for automation and synchronization purposes, based on the number of samples. The use of a robotic platform also allows the simultaneous processing of large numbers of samples, for example thirty-six 96-well plates. The automated method typically comprises: (a) reacting a compound of the invention with a transformed cell in the presence of L-PHA, and measuring the activity of alkaline phosphatase; and (b) comparing with a control in the absence of the compound, wherein an increase in alkaline phosphatase activity indicates that the compound has the ability to inhibit the processing of N-linked oligosaccharides. The transformed cells which can be used in The method of the invention includes the cell lines described herein or cell lines containing constitutive or inducible enzymatic activity such as the osteoblast cell lines. An alkaline phosphatase expression construct can be introduced into the cells to amplify the signal. The amount of proliferation of the cells is measured by determining the alkaline phosphatase activity. The alkaline phosphatase can be measured using conventional methods, for example, by using para-nitrophenyl phosphate as a substrate, and measuring its absorbance at about 405 nm. The conditions for carrying out the method will be selected when considering the nature of the compound and the cells used. For example, if the transformed cells are MDAY-D2 tumor cells, a concentration of about 1-6 x 103 cells, preferably 5 x 103 can be used. MDAY-D2 cells are generally cultured for about 10 to 30 hours, preferably 16 hours. at 20 hours followed by addition of L-PHA at a concentration of about 50 to 150 μg / ml, preferably 100 μg / ml. The alkaline phosphatase assay mixture may contain a buffer, for example diethanolamine buffer, and para-nitrophenyl phosphate at a concentration of about 1.5 to 4 mM, and preferably 2 to 3 mM, much more preferably 2.5 mM. The automated method of the invention can generally be used to identify compounds that antagonize as inhibitors of cell proliferation. For example, the method can be used to identify antagonists of cell growth inhibitors such as TGFβ or apoptotic agents such as TNFα. Therefore, the invention broadly contemplates a method comprising: (a) reacting a test compound with a transformed cell in the presence of a cell growth inhibitor; (b) measuring alkaline phosphatase activity; and (c) comparing with a control in the absence of the test compound, wherein an increase in alkaline phosphatase activity - indicates that the compound has the ability to antagonize with the cell growth inhibitor.

Properties of the Swainsonine Salts of the Invention The salts of the invention have useful pharmacological properties and provide antimicrobial properties, cancer suppressive, hemorrhoreactive, chemoprotective, radioprotective and immunomodulatory effects, and in particular can stimulate the TH1 arm of the cellular immune response. These properties are discussed in more detail in the following.

Cancer Suppression Properties The blocking of Golgi α-mannosidase II enzyme for carbohydrate processing prevents normal maturation of N-linked oligosaccharides in "complex-like" structures (Elbein, AD, Ann. Rev. Biochem 56: 497-534, 1987) which they are known to be important for the growth and metastatic spread of tumor cells (Dennis, JW Science 236: 582-585, 1987). In animal and tumor models, treatment with a Golgi mannosidase II inhibitor has been shown to inhibit the rate of tumor growth and metastasis (Dennis Cancer Res. 46.5131-5136, 1986. 1. Dennis, JW, Cancer Res. 50: 1867-1872, 1990. Newton, SAJ Nati, Cancer Inst. 81: 1024-1028, 1989). Golgi mannosidase II inhibitors such as swainsonin have cancer suppression properties in a wide variety of tumor types that include direct effects ^^ anti-metastatic and anti-invasion on tumor cells, and other anti-cancer activities such as immune stimulatory effects and myeloproliferative and hemorrhaging activities, as described herein.

Immunostimulatory Properties Blockade of the pathway in Golgi a-mannosidase II causes an accumulation of carbohydrate structure of "hybrid type", which have terminal residues. The exposed mañosa residues are an important feature directly related to immune stimulation (Sherblom, AP et al., J. Immunol., 143: 939-944, 1989, Yagita, M. and ^ 15 Saksela, Scand J. Immunun. 275-282, 1990). At the molecular level, it has been shown that certain cytokines, which include interferon (IFN), interleukin-2 (IL-2) and tissue necrosis factor (TFN-a) bind to carbohydrate structures that end up in mannitol structures such as those which accumulate when Golgi mannosidase II is blocked. These carbohydrate structures are found on cell surfaces and it is suggested that they improve cytokine binding to glycoproteins and cell surface receptors or co-receptors that are required to transmit the action of the cytokine within the cellular immune response. .

After infection with viral, bacterial or fungal pathogens, the immune response of the host involves inflammation and activation of the cellular and humoral arms of the immune system. CD4 + T cells can be stimulated to differentiate into helper T cells with the Thl phenotype which is associated with cellular immunity, or the Th2 phenotype, which is associated with the production of antibodies (Shindler, Annu Rev. Biochem 64: 621-651, 1995). Thl cells are characterized by the production of the cytokines INF-a, IL-2, TNFa, IL-12 while Th2 cells produce the cytokines IL-4 and IL-10. The Thl cytokines further promote the Thl response, while suppressing the Th2 response and, conversely, the Th2 cytokines promote the Th2 response and suppress the Thl response. The balance between Thl and Th2 responses is a major determinant of the outcome of infectious diseases, as well as autoimmunity and allergic reactions. It has been shown that the inhibition of Golgi α-mannosidase in mouse and in cell culture improves the immune responses mediated by Thl-dependent cells. This includes the activation of natural killer cells (NK) and killer cells activated by lymphokine (LAK) as well as the stimulation of T cells by antigens and IL-2 (Wall, KA, Proc. Nati. Acad. Sci. USA 85: 5644- 5648, 1988). Inhibition of Golgi α-mannosidase also improves tissue-mediated necrosis factor (TNFα) -dependent macrophage stimulation (Muchmore et al., Cancer Res. 50: 6285-6290, 1990) and IL-2-dependent stimulation of cells. LAK in vitro (Yagita et al., Scand, J. Immunol., 31: 275-282, 1990). In addition, inhibition of Golgi α-mannosidase improves the response of α-IFN, including antitumor and antiproliferative responses, as well as the induction of 2'-5 'oligoadenylate synthetase and the expression of TIMP genes (tissue inhibitors). metalloprotease) (Dennis, JNCI 81: 1028-1033, 1989, Korczak et al., Int. J. Cancer 53: 634-639, 1993). Cytokines bind to cell surface receptors and transmit signals to the nucleus through phosphorylation and dimerization of the family of signal transducers and transcription activators (STATs) of transcription factors. STATl is required for the antiviral response to a-IFN, for the Thl immune response and the associated production of cytokines, and for the clearance of the mouse hepatitis virus in vivo. Evidence for this is provided by the mutant STATl mouse agénico, which is normal in development, highly susceptible to infection by viral hepatitis and does not respond to IFN (Meraz, MA et al., Cell 84: 431-442, nineteen ninety six). The activation of STAT3 is associated with inflammation, notably the response dependent to IL-6. STAT6 is required for the Th2 response, as the agénic mutant mice are deficient in Th2 immune response (antibody dependent) and lack a normal IgG response to nematode infection. STAT4 is also required for the Thl response since mice deficient in this gene show a defect in the IL-12-dependent stimulation of NK and LAK cells, as well as in the production of Thl cytokines (Kaplan, Nature 382: 174-177, nineteen ninety six). 5 It has been demonstrated that the Thl cellular immune response is essential for the suppression of tumor growth and metastasis, and the elimination of certain viral, bacterial, fungal and parasitic infections, and in cancer. It has been shown that the importance of the Thl response for chronic viral infections including hepatitis B (Milich DR, Schodel F, Hughes JL, Jones JE, Peterson DL, 1997. J Virol 71: 3: 2192-2201), hepatitis C (Tsai SL, Liaw YF, Chen MH, Huang CY, Kuo GC, 1997. Hepatology 25: 2: 449-458), HIV (Clerci M. Shearer GM, 1994. Immuno Today 15.12: 575-581), labialis herpes simplex (Spruance SL, Evans TG, McKeough MN Thai L. Araneo BA, Dayens RA, Mishkin EM, Abramovitz AS, 1995. Antiviral Res 28: 1: 39-55), bacterial infections such as Pseudomonas aeruginosa infection of the respiratory tract in a rat model of cystic fibrosis (Johansen HK 1996, APMIS Suppl 63: 5- 20 42), leprosy caused by Mycobacterium leprae (Modlin RL 1994; J. Invest ~~ Dermatol 102: 6: 828-832), fungal infections which include Candida albicans (Romani L. et al., 1995; Immunol Res 14: 2: 148-162) and parasitic infections including Leishmania (Kemp M. 1997. APMIS Suppl 68, 1-33), and schistosomoasis, caused for one of the five species of flatworms known as schistosomes (Wynn TA et al., 1996, J. Immunol 157: 9: 4068-4078). Although interferon and interferon inducers have anticancer and antiviral activity, they appear to be insufficient in themselves to stimulate an appropriate Thl response capable of eliminating the disease. For example, interferons have been used in clinical trials for the treatment of most types of cancer, with variable efficacy (Goldstein D and Lasglo J, Can Res 46: 4315, 1986). It has also been shown that interferons have some efficacy in the treatment of hepatitis C and hepatitis B. In hepatitis, an initial response to a-IFN occurs in less than 50% of patients and in hepatitis C, 75-90% of all patients treated with α-IFN relapse (Hoofnagle JH et al., 1986 New Engl J Med: 315: 1575-1578; Davis GL et al., 1989 New Engl J Med; 321: 1501-1506). In addition, another Thl cytokine, IL-2, has been shown to be effective in the treatment of certain cancers, in patients with HIV and in cases of leprosy (Curr Opin Biotech 4: 6: 722-726, 1993).

Hemorrhoreactive properties / protection against radiation mortality and chemotherapy Myelosuppression is often a limiting feature of chemotherapy-dosing for numerous diseases including cancer (Hoagland, Hematologic Complications of Cancer Chemotherapy, In: The chemotherapy source book, Perry MC (ed) pp. 498-507, Williams & Wilkins: Baltmore 1992) and acquired immunodeficiency syndrome (AIDS) (McLeod and Hammer, Ann. Int. Med 117: 487, 1992, Richman et al, N Eng J Med 317: 192, 1987, Shaunak and Bartlett, Lancet 11:91, 1989; Walker et al, Clin Res 35: 435A, 1987). Patient support through periods of myelosuppression or decreased resistance in the infection is a critical part of the chemotherapeutic regimens. It has been shown that Golgi mannosidase II inhibitors (for example the free base of swainsonin) protect against the mortality of various chemotherapeutic agents (Orepide et al., 1991) as well as against lethal doses of irradiation (White et al. 3: 83-90; 1991). In these studies, increased survival in mice treated with swainsonin correlates with stimulation of bone marrow proliferation, bone marrow cellularity and grafting efficiency in mice (Orepide et al, 1991; White et al, 1991), as well as an improvement in peripheral blood counts.

Treatments using the swainsonine salts of the invention It is evident that the salts of the invention can be used in a method for the prevention, treatment and prophylaxis of tumor growth and tumor metastasis. The salts and compositions of the invention are especially useful in methods for the treatment of various forms of neoplasia such as leukaemias, lymphomas, melanomas, adenomas, sarcomas and solid tissue carcinomas in patients. In particular, the salts and compositions can be used to treat malignant melanoma, pancreatic cancer, cervical cancer, ovarian cancer, kidney cancer such as metastatic renal cell carcinoma, stomach cancer, lung, rectum, breast, bladder, gastric, hepatic, thyroid cancer, cancer of the head and neck such as head and neck cancers that can not be removed, cervical lymphangitic carcinomatosis, breast cancer, salivary glands, leg, tongue, lips, biliary duct, pelvis, mediastinum, urethra, oncogenic cancer , of the bladder, esophagus and colon and lung cancer of non-small cells, and Kaposi's sarcoma which is a form of cancer associated with patients infected with HIV in the acquired immunodeficiency syndrome (AIDS). The salts and compositions of the present invention can be used to treat immunocompromised subjects. For example, they can be used in a subject infected with HIV or other viruses or infectious agents including bacteria, fungi and parasites, in a subject undergoing bone marrow transplants and in subjects with chemically or tumorally induced immune suppression. The salts and compositions of the invention can be used as hemorrhaging agents and in particular to stimulate the proliferation of bone marrow cells in particular after chemotherapy or radiotherapy. The myeloproliferative activity of the salts and compositions of the invention can be determined by injecting the compound into mice, killing the mice, extracting bone marrow cells and measuring the ability of the compounds to stimulate bone marrow proliferation by directly counting bone marrow cells. bone and by measuring clonogenic progenitor cells in methylcellulose assays. The salts and compositions of the invention can also be used as antiviral agents in particular in membrane-enveloped viruses such as retroviruses, influenza viruses, cytomegalovirus and herpes viruses. The salts and compositions of the invention can also be used to treat bacterial, fungal and parasitic infections. The compounds of the invention can also be used in the treatment of inflammatory diseases such as rheumatoid arthritis and asthma. The compounds inhibit mannosidase and can turn the carbohydrate structures on neutrophils unable to bind to selectins. The selectins present in the damage site interact with the carbohydrate structures in the neutrophils in such a way that the neutrophils roll up along the epithelial wall, adhere, infiltrate and cause tissue damage. The salts of the invention have particular application in the prevention of tumular recurrence after surgery, that is, as an adjuvant therapy. It is evident from the properties of the salts of the invention that they can also be used to increase the anticancer effects of agents such as interleukin-2 and poly-IC, to increase the natural killer tumoricidal activity and of macrophages, to induce synthesis and secretion of cytokines, to improve the expression of specific antigens LAK and HLA class 1; to activate protein kinase C, stimulate the proliferation of bone marrow cells including the proliferation of hematopoietic progenitor cells and to increase grafting efficiency and the activity of the colony forming unit, to confer protection against chemotherapy and radiation therapy (for example of chemoprotective and radioprotective agents) and to accelerate the recovery of bone marrow cellularity, particularly when used in combination with chemical agents commonly used in the treatment of human diseases including cancer and acquired immunodeficiency syndrome (AIDS). For example, the salts of the invention can be used as chemoprotectants in combination with anticancer agents including doxorubicin, 5-fluorouracil, cyclophosphamide and methotrexate, and in combination with isonizide or with NSAID. The activity of the salts of the invention for a particular treatment application can be tested by various in vitro and in vivo models described herein and known in the art. In particular, antimetastatic effects of the salts and compositions of the invention can be demonstrated using a lung colonization assay. For example, melanoma cells treated with a compound can be injected into mice and the ability of melanoma cells to colonize the lungs of mice can be examined by counting the tumor nodules on the lung after death. The expression of tumor growth in mice by the compound administered orally or intravenously can be examined by measuring tumor volume. The cellular models and the animal models that make up the anticancer effects of the salts of the invention include the models set forth in Table 3. Examples of protocols for confirming the activities of the salts of the invention are included in the section of examples Other embodiments of the invention provide a method for treating a described condition which includes exposing a subject in need of such exposure to a pharmaceutically effective amount of a swainsonine salt of the invention, a metabolite of a described swainsonine salt, or a promedicamento or metabolic precursor of a metabolite thereof. In this embodiment, the metabolite can be used as an agent, for example, against a hepatitis C infection. A salt or composition of the invention can be used as a vaccine adjuvant to induce a potent immune response to itself and / or to induce immunity to antigens, particularly antigens that are usually poor immunogens. The salt or composition of the invention can increase the immunogenicity of the vaccine through the activation of antigen presenting cells, such as monocytes or macrophages, to release cytokines that can promote the help of T cells for B cells and the response of CTL. As a result, the salt or composition can induce a more favorable antibody response with high titers, which appear earlier in the course of immunization and persist over time, as well as an increase in memory and CTL responses restricted to CD8 + of CPH class I. A salt of the composition of the invention may be contained in a vaccine or may be administered separately. A salt of the invention can be used to improve the immunogenicity of antigens that induce T cell responses (e.g., T cell antigens), and in particular can be used to improve the immunogenicity of carbohydrate antigens associated with cancers or diseases. infectious Examples of vaccines which may use a salt or a composition of the invention to enhance immunogenicity include cancer vaccines (e.g. breast cancer vaccines) and vaccines for chronic infectious diseases.

EXAMPLES Example 1 Synthesis of swainsonine hydrochloride The free base of swainsonin (203.7 mg, 1.18 mmol) is dissolved in 4.0 ml of distilled water. Aqueous ÍM hydrochloric acid (1.41 ml, 1.2 equivalents) is added. After lyophilization, the amorphous residue is crystallized from methanol-ether or ethanol. Swainsonin hydrochloride (448.6 mg) is dissolved in 5.0 ml of methanol. After filtration, approximately 6.3 ml of diethyl ether are added dropwise over a period of 30 minutes with occasional stirring of the solution. The crystals begin to form after 0.25 ml of ether have been added. The crystallization solution is left at room temperature for 20 minutes. After filtering by suction and washing with 6 ml of 1: 2 of methanol: diethyl ether, colorless crystals are obtained (347.1 mg, 77.4% yield). This synthesis does not require chromatographic purification. The melting point of the clear crystal of swainsonine hydrochloride (prism) is 190-191 ° C. The solubility of swainsonine hydrochloride in distilled water at room temperature is about 3 g / ml, in contrast to the solubility of the swainsonine free base, which is about 0.8 g / ml (see table 5). 2 Synthesis of swainsonine hydrochloride The swainsonin hydrochloride can be synthesized from 1,2, -O-isopropylidene swainsonin. A 10% solution (w / v) of 1, 2-o-isopropylidene swainsonin in tetrahydrofuran, methanol, ethanol or isopropanol, is acidified by adding the same volume of 6M aqueous hydrochloric acid. After stirring overnight at room temperature, the solution is concentrated to dryness. The residue is dissolved in methanol or ethanol and decolorized with activated charcoal (50 ° C, 15 min). The activated carbon is separated by filtration and the residue is crystallized as described in example 1. 3 Solubility of Swainson's Hydrochloride Swainsonin free base samples synthesized using synthetic routes developed by Dr. David Dime (Toronto Research, Toronto, Ontario) and Dr. William Pearson (University of Michigan, Ann Arbor, Michigan), were recrystallized to obtain the hydrochloride salt, hydrobromide salt or swainsonine free base. The samples are weighed and exposed to the conditions indicated below. For the model of long-term stability or shelf life, various conditions were used to accelerate the decomposition process. Samples of crystalline prism swainsonine hydrochloride salt and swainsonine free base (a fluffy, white powder obtained from swainsonine hydrochloride is recrystallized from chloroform-methanol-diethylether) are weighed and exposed to the conditions described below (stressed samples). Unstressed samples are prepared at a concentration of 1 mg / ml and subjected to sextuplication chromatography in each run. After the indicated time interval, each tensioned sample is diluted with mobile phase to the same concentration as the unstressed sample. The remaining percentage of swainsonin hydrochloride or swainsonine free base is calculated based on the percentage of the hydrochloride or free base in the unstressed sample. Conditions include: (a) UV light for 7 days; (b) 105 ° C with atmospheric oxygen for 7 days (* = average of two samples); (c) 105 ° C under nitrogen for 7 days (* = average of two samples); (d) 70 ° C with low humidity for 7 days; and (e) 40 ° C with 75% relative humidity for 7 days. Other tests include: (f) UV light for 24 hours; (g) aqueous solution at 100 ° C for 2 hours; (h) aqueous acid treatment for 24 hours; (i) aqueous alkaline treatment for 4 hours; and (j) 3% hydrogen peroxide (aqueous) for 4 hours. Surprisingly, the thermal stability of the hydrochloride salt is greater than that of the free base or the hydrobromide salt (see table 4). further, the photochemical stability of the hydrochloride salt is significantly higher than that of the hydrobromide salt (see table 4). Table 5 shows the physical properties of swainsonine hydrochloride compared to the free base and swainsonin hydrobromide, and swainsonin hydrofluoride. Swainsonin hydrochloride, hydrobromide and free base are exposed at 50 ° C / 50% relative humidity (RH) and 80 ° C / ambient humidity for 4 weeks. At the baseline, and at intervals of 1 week, the stability of the test materials is measured by CLAR, as in the previous. In addition, an evaluation of color and moisture is made at the beginning and at the end of the study, and the samples of the base and salts are weighed, and the coloring and formation of water are also recorded.

The 4 Synthesis of swainsonine hydrobromide The free base of swainsonin (299.7 mg) is dissolved in distilled water (6.5 ml). Aqueous hydrobromic acid 1 M is added (1.1 equivalents) and the solution is lyophilized. The residue is crystallized from methanol-diethylether in a manner similar to that of the hydrochloride salt in example 1. The salt of swainsonin hydrobromide (341 mg, 77.6%) is obtained as colorless crystals with a melting point of 153 - 15 ° C.

Example 5 Synthesis of swainsonine hydrofluoride The free base of swainsonin (301.03 mg) is dissolved in methanol (10 ml) and a solution of 48% hydrogen fluoride (84 microliters) is added. After concentrating the solution, the residue is crystallized from boiling methanol. Colorless needles are obtained (14.9 mg, 4.5%). These needles decompose at a temperature above 152 ° C without melting.

Example 6 X-ray crystallographic analysis of swainsonine hydrochloride and swainsonine hydrobromide An X-ray crystallographic analysis is carried out using conventional procedures. Space groups and cell parameters are determined from photographs with a precision camera. The refined cell parameters are obtained by diffractometer measurements of 12 high angle reflections (40 ° < 2? < 60 °) and application of the least squares method. Tables 1 and 2 provide the crystallographic data. The glass dimensions are chosen for data collection in a diffractometer, using copper radiation Ka and a scanning mode? 2? with a scanning speed of 2o / min. Three standard reflections, monitored every 100 reflections, show only variations of random intensity within 5%. The intensities were corrected for Lorentz and the polarization factors. Absorption corrections were not applied. The crystal structures were determined by direct methods using a program such as the SHELXS-86 program or the SIR-88 program. In each case, map E shows the portions of all atoms that are not hydrogen in the structures. The refinement of the structure and the electronic difference density calculations do not show residual electronic density. The final discrepancy factors converge at R = 3.6% at 2s per ~ 1200 intensity data (hydrochloride salt) and R = 3.8% at 2s per 1002 intensity data (hydrobromide salt). The three-dimensional structure of the swainsonine hydrochloride salt is shown in Figure 1, while the structure of the swainsonine hydrobromide salt is shown in Figure 2. Figures 3 and 4 are crystal packing diagrams for swainsonin hydrochloride and swainsonin hydrobromide, respectively. The unit cell lengths for the hydrochloride salt are a = 8.086 ± 0.01, b = 9.386 + 0.01, c = 13.621 ± 0.01Á. The unit cell is orthorhombic (all angles = 90 °) and a space group is P212121. The atomic coordinates for the salt are shown in table 1. The final discrepancy factor R = 3.6% at 2s for approximately 1200 intensity data. Some torsion angles are as follows: H7-C7-C8-H8 39.75 ± 3.33; H8-C8-C9-H9J. -140.68 ° + 3.10; H8-C8-C9-H92 -20.90 ° + 2.86. The best least squares planes of the SW hydrochloride salt and SW diacetate are set forth in Table 6. From Table 6, it is evident that the 4 atoms in swainsonin diacetate are marginally flatter than in the structure of swainsonin hydrochloride crystal. The molecules of swainsonine hydrochloride in the unit cell are held together by hydrogen bonding interaction between the protonated N atom and three hydroxyl oxygen atoms of one molecule to the chloride ions of other molecules. The binding distances are as follows: Nl-Hl = .88Á, from Hl to the chloride ion, 2.35 Á; O5-H50 = 0.78; from H50 to the chloride ion 2.33 A; O7-H70 = 0.74 A; from H70 to chloride ion 2.44 Á; 08 to H80 = 0.67 Á; and from H80 to the chloride ion 2.50 Á. The unit cell lengths for the hydrobromide salt are a = 8.405 ± 0.01, b = 8.629 + 0.01, c = 14.118 + 0.01Á. The unit cell is orthorhombic (all angles = 90 °), and the space group is P212121. Table 2 shows the atomic coordinates for the salt. The final discrepancy factor R = 3.8% at 2s for approximately 1200 intensity data. Some torsion angles are the following: H7-C7-C8-H8 40.07 ± 0.21; H8-C8 -C9-H9- ,. -137.29 ° ± 0.09; H9-C8-C9-H92 -16.96 ° +, 0.19. The best squared minimum planes of the SW hydrochloride salt and SW diacetate are set out in Table 6. The swainsonin hydrobromide molecules in the unit cell are held together by hydrogen bonding interactions between the three oxygen atoms of hydroxyl of a first molecule with the bromide ions of other molecules, and the protonated N atom of the first molecule with an oxygen atom on a second molecule. The bonding distances are as follows: Nl-H = 0.91Á; from H to the oxygen atom 08 1.94 A; 05 -H50 = 0.82 Á; from H50 to the bromide ion 2.47 Á; O7-H70 = 0.82 A; from H70 to the bromide ion 2.61 A; 08 to H = 0.82 Á; and, from H to the bromide ion 2.56 Á. 5 The main significant difference between the crystal structures of the hydrochloride and hydrobromide salts is in the intermolecular hydrogen binding scheme. In swainsonine hydrochloride, each ion Cl "is the acceptor for 4 hydrogen bonds, from? -HCl; 05-H ... C1; 07-H Cl; 08-H Cl. In swainsonine hydrobromide, the Br ion occupies a different position with respect to the swainsonin molecule and is an acceptor for 3 H-bonds from 05-H ... Br; 07-H ... Br;; 08 -H ... Br and the nitrogen-H bond is at 08, that is,? -H ... 08.15 MR spectrum? of swainsonin and swainsonine hydrochloride We analyzed the -I and 13C RM spectra? of samples of swainsonin and swainsonin hydrochloride by comparison with data reported for swainsonin (M. J. Schneider, et al., Tetrahedron 39:29, 1983). The compounds used in the study were dissolved in D20 (Isotec, Inc.) to a concentration of about 4.5 mg / ml. The chemical shift of -? which is reported in table 7 is confirmed by COZY and by experiments of -I-13C HSQC. The differentiation of the axial and equatorial protons in the six-member ring is obtained by examining the neighborhood coupling constants and the general observation that the axial protons in the six-member rings are usually covered in relation to the equatorial protons (FA Bovey, Nuclear Magnetic Resonance Spectroscopy, Academic Press, New York 1988). Methylene protons at C-3 on the five-membered ring are assigned pseudo-axial and pseudo-equatorial positions by the 2-D ROSEY experiment (which provides data similar to a 2-D NOE spectrum but which creates inter-space correlations between protons by rotating frame mechanism N03, A. Bax and DG Davies, J. Magn. Reson 63: 207, 1985, D. Heuhaus and M. Williamson, The Nuclear Overhauser Effect in Structural and Conformational Analysis. ., New York, 1989, and WE Hull in Two-Dimensional NMR Spectroscopy-Applications for Chemists and Biochemists, 2nd Edition, Edited by WR Croasmun and R, MK Carlson, VCH Publishers Inc. New York, 1994, Ch.2). The methylene protons C-3 (2,754 and 2,420 ppm) appear as part AB of an ABX spin system with H-2 (4,217 ppm). This assignment is also supported by a larger neighborhood coupling constant with H-2 of 7.9 Hz, since the dihedral can be less than 60 ° between H-2 and H-3. Coupling constants are reported only for those multiplets which show a well resolved division. The equatorial protons of the six-member ring appear as extended unresolved multiplets due to the superposition of several small coupling interactions. The chemical shifts 13C (see Table 9) were confirmed by the modulated spin class J and the HSQC _I-13C spectra. When the effects of substituents on the six-membered ring are taken into consideration with the chemical shifts for C-5 and C-6 which agree with the model of perhydroindolizine compound (HO Kalinowski, S. Berger and S. Braun, Crbon- 13 NMR Spectroscopy, J. Wiley and Sons, New York, 1988). The same procedures were used to assign NMR spectra of swainsonin hydrochloride. An initial examination of the NMR spectra indicates a loss of protection of all chemical shifts relative to the SW sample (see Table 7). The protons in C-3, C-5 and C-9 were the most affected by the protonation of nitrogen. Most of the chemical shift assignments for swainsonin hydrochloride (SWHC1) can be made by comparison with the SW data, however, COSY and ROESY spectra are required to confirm the assignments particularly of the C-3 protons. In the sample, SWHC1 there is an inversion of the order of the chemical shifts of pseudoaxial and pseudo-equatorial C-3 protons. The pseudoaxial C-3 proton has a higher frequency in SWHC1 (3,379 ppm) in relation to the pseudo-equatorial C-3 proton (3,306 ppm). This assignment is confirmed by the ROESY data where the multiplet of 3,379 ppm shows to be clearly separated through the spatial correlations with the axial protons in C-9 (2,959 ppm) and C-5 (2,805 ppm). The neighborhood coupling constants between protons C-3 and H-2 support these assignments (see table 8). The carbon chemical shifts were assigned from the modulated spin class J and the -I-13C HSQC spectra. With the exception of C-5, all the carbon resonances of SWHC1 were covered by variable amounts in relation to SW (see table 9). This is generally observed when the alkylamines undergo protonation (H.O. Kalinowski, 1988, supra). The protection experienced by the carbons 6 and 8 of the six-membered ring can also be attributed in a small part to the introduction of an axial hydrogen in the nitrogen. The axial N-H can create 1.3 diaxial steric interactions with the axial protons C-6 and C-8 resulting in a substituent effect? on the chemical shifts 13C of C-6 and C-8 (H.O. Kalinowski, 1988, supra). The chemical shift examination and the ROESY data indicate that there is no significant difference in the general structures of these samples. The protonation of nitrogen seems to occur with the proton N-H occupying an axial geometry. However, the protonation of nitrogen need not have made the ring conformations more rigid and adopt the structure shown below: This conclusion arises from the observation of a coupling of five unions of 0.7 Hz between H-1 and H-5e. The spin decoupling experiments confirm this coupling interaction. Long range couplings of this type are highly stereospecific and require that all atoms in the coupling path be in a zigzag or "W" coplanar type structure. The conformation of five-member rings is generally more flexible even in large structures such as steroids. Therefore, protonation must fix the geometry of the atoms in the long-range coupling pathway as shown, in order to produce the division observed on the multiplets H-1 and H-5e. The stiffer structure may also justify changes in the neighborhood coupling constants in the 5-member ring in the SWHCl sample (see Table 8). In conclusion, the chemical shifts of _I and 13C of swainsonin hydrochloride and its protonated nitrogen analogue have been fully assigned and most of the coupling constants _! -! they have been reported for the well-resolved multipletes. The most significant structural difference between the two samples was the more rigid conformation of the SWHCl molecule as indicated by the long-range spin-I coupling interactions. 8 Preformulation studies The preformulation studies of swainsonine hydrochloride, drug substance in volume, and in combination with powder and semisolid filled gelatin capsules are carried out with respect to the following: hygroscopicity, pH, stability and solubility. It is found that the compound is highly hygroscopic. The studies carried out on the medicine in volume show that the compound absorbs approximately 8% (w / w) and 24% (w / w) of water in the first 2 and 8 hours, respectively, at 75% of RH and becomes a semi-solid At 20% and 50% of RH, it absorbs 1.9% and 2.1% of water, by the Karl Fischer method, after 48 hours of storage. Moisture uptake decreases when anhydrous excipients (for example anhydrous lactose and mannitol powder) are used to formulate the pharmaceutical active substance in a hard capsule. The compound is highly soluble in aqueous and hydrophilic vehicles. Therefore, for soft gelatin capsule formulations, hydrophilic carriers are preferred. The use of a cosolvent such as glycerin or propylene glycol in the PEGs may be feasible for liquid or semi-solid fillings. The results of a pH study show that the compound is stable in buffered solutions at pH 4 and 7 under environmental and stress storage conditions (40 ° C and 50 ° C).Example 9 NMR of a drug substance in volume of swainsonine hydrochloride Proton nuclear magnetic resonance (NMR) and homonuclear correlation spectroscopy (COZY) spectra are obtained for the hydrochloride salt of (-) (SS, 2S, 8R, 8aR) -1, 2, 8-trihydroxyoctahydro-indolizidine (swainsonine hydrochloride, a white to off-white crystalline solid, molecular weight 209.66, pKa, 7.4, melting range 189-190 ° C) in deuterated water (D20). D20 is also used as an internal reference at 4.60 ppm. The peak assignments are based on the NMR spectra of the proton and the COZY spectral couplings, determined in Example 7. The differentiation of axial and equatorial protons is obtained by examining the neighborhood coupling constants and the general observation that in the six-member rings, the axial protons are usually covered in relation to the equatorial protons. The methylene protons in C-3 are assigned to pseudoaxial and pseudo-equatorial positions by 2-D ROSEY experiments. The carbon NMR spectra, the proton annex (APT) tests and the heteronuclear spin tale coherence spectra (HSQC) are obtained for the hydrochloride salt of (-) - (lS, 2S, 8R, 8aR) - 1,2,8 - trihydroxyoctahydro - indoli z idina (swainsonine hydrochloride, white to off-white crystalline solid, molecular weight 209.66, pKa 7.4, melting range 189-190 ° C) in D20. The peak assignments are based on the carbon NMR spectra, the DEPT and the spectral interpretation of HSQC are shown in Table 10. The assignments are based on spectral information found in Nakanishi, K., One-dimensional NMR Spectra by Modern Pulse Techniques, University Science Books, Tokyo, Japan, 1990.

Example 10 Quantitative macroanalysis Elemental microanalysis (CHN) was performed in a hydrochloride salt of (-) (SS, 2S, 8R, 8aR) -1, 2, 8-trihydroxyoctahydro-indolizidine (swainsonin hydrochloride, a white to off-white crystalline solid, molecular weight 209.66 , pKa 7.4, melting range 189-190 ° C) using a Perkin Elmer 2400 combustion analyzer. Chlorine analysis was carried out by potentiometric titration. Table 11 shows the results.

Example 11 Infrared absorption spectrum The infrared spectrum of the Fourier transform (FTIR) of the hydrochloride salt of (-) (SS, 2S, 8R, 8aR) -1, 2, 8-trihydroxyoctahydro-indolizidine (swainsonin hydrochloride, a white to off-white crystalline solid, molecular weight 209.66, pKa 7.4, melting range 189-190 ° C) is taken in a granule which is obtained. The main absorption bands are consistent with the structure for the compound, and in Table 12 the assignments of the characteristic absorption bands are included. These assignments are based on spectral information found in Silverstein, R.M. , Bassler, G.C., and Morrill, T.C. Spectrometric Identification of Organic Compounds, 3nd, ed. , John Wiley & Sons, New York, 1974, chapter 3 and in Introduction to Spectroscopy, by Pavia D.L., Lampman, G.M. and Kriz, G.S., Saunders Golden Sunburst Series Chapter 2.

Example 12 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of the hydrochloride salt of (-) (SS, 2S, 8R, 8aR) -1, 2, 8-trihydroxyoctahydro-indolizidine (swainsonin hydrochloride, a white to off-white crystalline solid, molecular weight 209.66, pKa 7.4 , melting range 189-190 ° C) shows no absorption peaks in the UV region examined from 200 nm to 300 nm by the evaluation of peak purity by CLAR.

Example 13 Mass spectrometry The hydrochloride salt of (-) (SS, 2S, 8R, 8aR) -1, 2, 8-trihydroxyoctahydro-indolizidine (swainsonin hydrochloride, a white to off-white crystalline solid, molecular weight 209.66, pKa 7.4, melting range 189- 190 ° C) is characterized by chemical ionization (Cl) (methane) mass spectrometry in a high resolution dual focus magnetic field instrument VG ZAB IS. Figure 5 shows the spectrum and the fragmentation scheme shown in Table 13.

Example 14 X-ray powder diffraction of dry swainsonine hydrochloride for formulations We show that a dry sample of swainsonin hydrochloride is crystallographically similar to the drug substance in original volume. X-ray powder diffraction studies show that a zero-core sample assembly technique provides a reproducible characteristic dust pattern for the drug.

Example 15 Thermal analysis The differential scanning calorimetry (DSC) thermogram for the hydrochloride salt of (-) (SS, 2S, 8R, 8aR) - 1, 2, 8 - trihydroxyoctahydro-indolizidine (swainsonin hydrochloride, a white to off-white crystalline solid, molecular weight 209.66, pKa 7.4, melting range 189-190 ° C) shows a melting endotherm from about 187.5-190.3 ° C when heated to 5 ° C / min under a nitrogen purge of 45 ml / min. The thermogravimetric analysis (TGA) shows a weight loss of approximately 0.20% at 160 ° C and a melting endotherm from 187.6-190.5 ° C when heated at 5 ° C / minute under a nitrogen purge of 40 ml / min.

Example 16 High resolution liquid chromatography (HPLC) The reversed-phase isocratic high-resolution liquid chromatography (HPLC) method is developed to test both the potency and related substances of the drug substance. The quantification of the drug substance is carried out by comparison with an external standard of the substance. Related substances are quantified by area percent. The chromatographic process for related potency and substance separates the drug substance from its synthetic precursors and potential impurities. The relevant chromatographic conditions for the CLAR are the following: column: Prodigy 5μ ODS-2 (25 cm x 4.6 mm DI); Mobile phase: acetonitrile buffer (10 mM KH2P04, pH = 9.0) 5:95; Flow rate: 1.0 ml / minute; Injection volume; 10 μl; Detection: UV, 205 nm; Room temperature; Sample concentration: 1.0 mg / ml; Diluting sample: Mobile phase. In Figure 6 a representative chromatogram is shown.

Example 17 Method to determine the inhibition of Gol II mannosidase II and lysosomal in vitro The swainsonin test compound is prepared by serial dilution 0.4 of a 40 μM concentrate. In each determination, 10 μl of diluted test compound, 25 μl of 10 mM para-nitrophenyl-amyranoside, 200 mM sodium acetate are present. pH 5.6 and 15 μl of Golgi mannosidase II from purified rat liver. After incubating the reaction for 60 minutes at 37 ° C, the reaction is suspended with 50 μl of 0.5 M sodium carbonate. The absorption is read at 405 nm. After subtracting the blank from the positive controls and samples, the samples are normalized against the positive control measure using a variable-pitch slanted curve adjustment, with background = 0, upper part = 100. The signal is proportional to the amount of products from the reaction not inhibited. The CIS0 calculated for Golgi mannosidase II inhibition purified by swainsonin hydrochloride is 0.021 μM.

The effects of the compounds of the invention on lysosomal mannosidase were measured by adding (10 μl) of the compounds in 96-well ELISA plates followed by the addition of 200 mM sodium acetate pH 5.0 and 25 μl of p-nitrophenyl aD-mannopyranoside 10 mM. 15 μl of lysosomal mannosidase (approximately 8 mM / ml) are added to each well and the plates are incubated for 60 min at 37 ° C. The reaction is stopped by the addition of 50 μl of 0.5 M sodium carbonate and the formation of p-nitrophenol with a plate adjusted to 405 is measured. The CIS0 calculated for the inhibition of lysosomal mannosidase by swainsonine hydrochloride is 0.045 +, 0.010 μM.

Example 18 A. Assay of L-PHA cells to measure the inhibition of mannosidase II in cells The swainsonin hydrochloride test compound is prepared by serial dilution 0.5 of a 40 μM concentrate in 50 μl of fetal bovine serum (FBS) 5% in minimal essential medium (MEM). To 50 μl of test samples diluted in 96-well plates, 10,000 MDAY-D2 tumor cells are added in 50 μl of FBS 5% in MEM, to each well. The samples are incubated at 37 ° C overnight in a C02 5% incubator. The test wells are prepared in duplicate by the addition of 25 μl / well of 5% FBS in MEM or 5% FBS in MEM containing 100 μg / ml of L-PHA. The samples are incubated again at 37 ° C overnight in a 5% C02 incubator. The viability and / or proliferation of the cells in each well is measured using phenazine methylisulfate (PMS) and the salt of (3 (4,5-dimethylthiazol-2-yl-5- (3-carboxymethoxyphenyl) -2,4- sulfofenil) -2H tetrazolium ("MTS") as described in the Promega CellTiter 96 AQ instructions The absorption is read at 490 nm The loss of toxicity of L-PHA is directly related to the entry of the drug into the cells and with the inhibition of Golgi mannosidase II, and the loss of the carbohydrate structures that bind to L-PHA on the surfaces of the cells.

B. High resolution L-PHA assay Materials and methods Chemical substances . L-PHA, Triton X-100 and para-nitrophenyl phosphate are obtained from Sigma; Diethanolamine is purchased from Fisher.

Cells: The origin and properties of the reticular lymphoid tumor of the DBA-2 strain of MDAY-D2 have been previously described (Kerbel, RS, Florian, M. Man, MS, Dennis, J and McKenzie IF (1980) J. Nati. Cancer Inst. , 64, 1221-1230). The cells are cultured in modified Eagle's medium containing 2% heat inactivated fetal bovine serum (Gibco BRL) at 37 ° C in a humidified atmosphere of 95% 02/5% C02.

Alkaline phosphatase assay. Determinations were made using 96-well plates. Each well contains a variable number of MDAY-D2 cells maintained in 125 μl of culture medium supplemented with 2% fetal bovine serum. The reaction with alkaline phosphatase is initiated by adding 75 μl of assay mixture (1 M diethanolamine buffer, pH 9.8, 2 mM MgCl 2, 1% Triton X-100 and 2.5 mM para-nitrophenylphosphate) and incubated at 37 ° C until for 90 minutes. The reaction is stopped with 80 μl of 3.5 M NAOH. After 15-30 min of color development, the absorbance of the chromogenic product para-nitrophenol at 405 nm is measured using a multi-well scanning photometer (Thermomax Multiplate Reader, Molecular Devices). Background values were determined by assays performed in a culture medium only in the absence of cells and were subtracted in a usual manner. The linearity between the absorbance at 405 nm and the concentration of para-nitrophenol in the range of 0.2.5 (e = 17.23 mM ^ cm-1).

Analysis by means of L-PHA tests. The procedure is completely automated through the use of a robotic workstation (Biomek 2000, Beckman) capable of processing nine 96-well plates simultaneously. The determinations were made in 96-well flat bottom plates (88 samples + 8 controls per plate). Each well (columns 1-11) receives 10 μl of compound (in DMSO 2.5%), while 10 μl of 2.5% DMSO in water is added to column 12. All of the 96 wells received 5 x 103 MDAY- cells. D2 in 90 μl of culture medium supplemented with 2% FCS. After 16-20 h of incubation at 37 ° C, 25 μl of L-PHA (100 μg / ml in culture medium) is added to the first 11 columns and to 4 wells of the number 12 (positive control). The other 4 wells received 25 μl of medium supplemented with 2% FCS (negative control). The assay plates are maintained for 30-36 h at 37 ° C and the alkaline phosphatase activity is measured according to the protocol described above using an incubation time of 1 h. Cell density is subconfluent through the course of the assay. The proliferation indexes are expressed as percentage values, calculated with the formula: Normalized signal = (sample A405 - mean of A40S of positive control / average of A405 of negative control - mean of A405 of positive control) The CIS0 calculated from inhibition of Golgi mannosidase II by swainsonin hydrochloride in cells is 0.057 + 0.01 μM.

Example 19 Effect of swainsonin hydrochloride on the proliferation of SP1.A3, a mammary tumor cell, in vitro proliferation The TGFßl and TNFα cytokines affect cell growth, lymphoid cell activation, tissue differentiation and cell death by apoptosis. When these cytokines induce cell growth, the differentiation death however is highly specific for the type of cell and tightly regulated during normal differentiation. Mitogenic effects of TGFα and TNFα have been reported for melanoma, colon carcinoma and ovarian cancer. The proliferation measured by the growth factor can be induced directly through its signaling pathway or by improvement of another expression of the growth factor receptor. Mouse SP1A3A mammary carcinoma cells are grown for 24 hours in culture medium containing 10% bovine serum with and without swainsonin hydrochloride at a concentration of 0.2 μg / ml. In the next 24 hours, the cells are maintained in a serum-free medium (SFM) with and without swainsonin hydrochloride. The cells are then grown in media of growth factors for 6 hours or exposed to one of the following growth factors: TNFa (tumor necrosis factor-a), TGFβ1 (transforming growth factor-β), TGFa, platelet-derived growth factor (PDGF9, epidermal growth factor (EGF). Tritiated thymidine is added during the final 18 hours, cells are harvested using a multi-cell harvester and radioactivity is measured with a β-counter as a measure of Cell proliferation As shown in Figure 7, proliferation of SPl.A3a cells is stimulated by growth factors TGF-ßl and TNF-a, and treatment with swainsonin hydrochloride suppresses TGF-ßl-dependent growth stimulation and TNF-a.

Example 20 Anticancer activity of swainsonine hydrochloride in vivo A. Effects of swainsonin hydrochloride on the growth of tumor cells SPl .A3a in mice A metastatic subclone of the SPI tumor line (A3a) of mouse mammary adenocarcinoma is maintained in exponential growth in RPMI 1640 containing 10% FBS. Cells are harvested and resuspended at 1 x 10ß / ml or 1 x 107 / ml in PBS and 0.1 ml containing 1 x 105 injected S.C. on the right flank of 7-week-old female CBA / J mice (Jackson Laboratories).

Alzet miniosmotic pumps are implanted subcutaneously, on the opposite side of the anesthetized animals. The pumps are sebated to supply saline (control) or 0.5 mg / kg / day of swainsonine hydrochloride for 28 days. The mice are monitored to determine the appearance of a palpable tumor and the subsequent tumor growth is measured using bernier calipers. The weights of the tumors and the number of lung metastases are measured on day 42. The mean volume of tumor on days 32, 39 and 42 of treatment are higher in the control animals (-) than in the mice that received hydrochloride of swainsonine by means of osmotic pumps for 28 days (+) (see Figure 8). The difference in the mean volume of tumor between the control groups and the group subjected to swainsonin hydrochloride on days 32, 29 and 42 of treatment are 35%, 27% and 32%, respectively. The mean tumor weight determined on day 42, at the slaughter point for the 5 animals in the control group is higher than that of the 4 animals in the group with swainsonin hydrochloride and is 7.35 g versus 4.87, respectively. The treated group has only one very large tumor. At the point on day 42 of sacrifice, the incidence of lung metastasis in control mice is on average 1.8 nodules / mouse and an average of 0.25 nodules / mouse in mice treated with swainsonin hydrochloride.

This experiment confirms the antitumor activity of the hydrochloride salt of swainsonin. In fact, the dose used is 8 times less than that used in the initial experiment performed with the swainsonine free base.

B. Effects of oral swainsonin hydrochloride in drinking water on the growth of tumor cells SPl .A3a in mice The experiment is repeated using swainsonine hydrochloride administered in drinking water. The mouse mammary adenocarcinoma SPl.A3a is maintained in exponential growth in RPMI 1640 medium containing 10% FBS. The cells are harvested and resuspended at 3 x 105 ml in PBS and 0.1 ml containing 3 x 10 4 cells injected S.C. on the right flank of 7-week-old female CBA / J mice (Jackson Laboratories) (n = 25). The mice are subsequently supplied with water to drink alone (n = 13) or with drinking water containing 10 μg / ml swainsonin hydrochloride (n = 12) (equivalent to a dose of 2 mg / kg / day). Once the palpable tumor is evident, the size of the tumor is measured twice a week using vernier calipers. At the end of the treatment period, the tumors are removed and weighed. Mice displaying tumors with 10 μg / ml swainsonine hydrochloride in drinking water have smaller growth tumors compared to control mice treated only with water. In Figure 9 the results are shown. The mean tumor size is much smaller for mice treated with swainsonin hydrochloride (+) compared to mice treated with saline (-). These differences are statistically significant for the time points shown. The mean tumor weights at 31 days in the treated groups are 1.79 g and in those not treated with 3.33 g. In conclusion, the experiments demonstrate that swainsonine hydrochloride has anticancer activity in vitro and in vivo. In addition, anticancer activity is demonstrated using a much lower dose than previously reported for the swainsonine free base.

Example 21 In vitro effect of swainsonine hydrochloride and swainsonin in murine bone marrow progenitor cells (CFU-E and CFU-GM).

MATERIALS AND METHODS Animals Female pathogen free C57BL / 6 rats, 8-9 weeks old, obtained from Jackson Laboratories were used. The environment of the room and the light period were controlled: 24 ° C; humidity 50 ± 20%; 12 h of light and 12 h of darkness. The mice were housed one per cage with ad libitum access to a granulated commercial laboratory diet and to sterile (autoclaved) tap water. materials Swainsonin hydrochloride is manufactured by Seres Laboratories, CA. FBS and methylcellulose (MethoCult M3330) are purchased from Stem Cell Technologies, Inc. (Vancouver, BC). Dulbecco's medium modified by Iscove is prepared using Gibco BRL spray medium, deionized water and sterilization by filtration. For the handling of the cells, the medium is supplemented with 2% FBS and 50 μM β-mercaptoethanol (referred to as IMDM / FBS).

Cell harvest Healthy mice treated with GD0039 are sacrificed by asphyxia by C02. Bone marrow (BM) cell suspensions are prepared under sterile conditions by washing both femurs and tibiae with IMDM / FBS using a 26 gauge needle. Single cell suspensions were made at 10 .. O ml of IMDM / FBS. The concentration of nucleated cells in each suspension is determined by triplicate beads in a hemocytometer. A portion of the cells is further divided into medium at the appropriate concentration before being plated for parental testing.

Progenitor cell assay The colony-forming units (ufe) were estimated by the methylcellulose method. Plates were plated in triplicate 1 ml suspensions containing 2 x 10s BM nucleated cells in 0.1 ml IMDM and 0.9 ml MethoCult (M3330), in 35 mm tissue culture boxes. Swainsonine hydrochloride or swainsonine was added to certain plates, in 0.1 ml of IMDM at concentrations of 30 μg / ml and 3 μg / ml, which provides final concentrations of 3 μg / ml. The MethoCult medium (M3330) contains 30% FBS and 10 ng / ml erythropoietin and is designed for the growth of early erythroid progenitor cells (CFU-E), which are classified after 3 days of incubation at 37 ° C in a humidified atmosphere containing 5% C02. For granulocyte-macrophage progenitor cells (CFU-GM), MethoCult M3230 contains 30% FBS, does not contain any additional growth factor and supports the growth of (CFU-GM) which are classified after 7 days of incubation at 37 ° C in a humidified atmosphere containing C02 5%. Some SCF and / or GM / CSF plates are added in 0.1 ml of IMDM to provide a final concentration of 50 ng / ml or 5 ng / ml (ED50) for SCF and 0.25 μg / ml or 1.7 μg / ml for GM- CSF Colonies containing more than 20 cells are classified using an inverted microscope with an optimal brightfield system and an enlargement of 40x or lOOx.

Results The BM cells of a healthy mouse and a mouse dosed with 20 μg / day of swainsonin hydrochloride for 4 days were analyzed in a CFU assay using M3330 methylcellulose. Both swainsonin hydrochloride and swainsonin significantly increase the amount of early CFU-E, counted on day 3, when added to methyl cellulose in vi tro (see Table 14). The high (3 μg / ml) and low (0.3 μg / ml) concentrations of swainsonin hydrochloride and swainsonin stimulate the number of CFU-E to the same extent when added to control (untreated) control BM cells. This is a dose dependent effect when BM is used for mice treated with swainsonine hydrate in vivo. Both swainsonin hydrochloride and swainsonin stimulate erythroid progenitor cells in vitro at approximately the same rate. At concentrations from 0.03 μg / ml to 10 μg / ml, they cause a ~ 3-fold increase in the amount of early CFU-E. Both swainsonin hydrochloride and swainsonin also stimulate granulocyte-macrophage progenitor cells in vitro (see Figure 11) BM cells from a healthy mouse C57BL / 6 are seeded onto plates in 1.0 ml suspensions obtained from a mixture of 0.8 ml of methylcellulose M3230, 0.1 ml of cell suspension, 0.1 ml of SWHCl and 0.1 ml of cytosine: 1-SCF 50 ng / ml, 2-SCF 5 ng / ml, 3-GM-CSF 1.7 μg / ml, 4 - GM-CSF 0.25 μg / ml, 5 - SCF 50 ng / ml + GM-CSF 0.25 μg / ml, 6 - without cytosines). In the absence of specific stimulating factors, swainsonine hydrochloride shows an increase of ~ 4 fold in CFU-GM.

Example 22 Toxicology and pharmacokinetic studies Pharmacological and toxicological studies were carried out with swainsonine hydrochloride. In particular, the following was investigated: (a) the pharmacokinetics of the compound in rats and monkeys; (b) acute toxicity at significant multiples of the proposed human dose; (c) the toxicity profile of the compound is compared to the profile of the literature for the swainsonine free base; (d) potential for genotoxicity; (e) course in time, dose dependence, tissue sensitivity and reversibility of oligosaccharide accumulation in tissue; and (f) Serum AST and relationship with liver histology. Studies indicate that acute toxicity to swainsonin occurs only at very high doses, 13,000 times the proposed human dose. Chronic studies indicate that the thyroid and possibly the kidney would be the sites of reversible accumulation of oligomanosides in lysosomes at the doses proposed for humans.

Example 23 Representative protocols in vivo and in vi tro A. Administration of swainsonine hydrochloride for the inhibition of pulmonary metastasis B16F10 melanoma tumor cells are cultured for 48 hours in the presence or absence of swainsonin hydrochloride (0.36 μg / ml) before the injection of 105 cells into the lateral tail veins of C57BL mice. Pulmonary nodules are counted on day 24 after injection of tumor cells as described in Dennis, JW, Cancer Res. 46: 5131-5136, 1986.

B. Swainsonine hydrochloride for the inhibition of colonization by tumor cells of the lung The mice are given water to drink with or without 5.0 μg / ml swainsonine hydrochloride 2 days before the tumor cells are injected into the lateral tail vein and maintained on a swainsonine hydrochloride regimen for periods of 1-17 days Lung nodules are counted on day 24 after injection of the tumor cells.

C. Inhibition of human tumor growth in mice Athymic nude mice injected subcutaneously with MeWo, a human melanoma tumor cell line, are treated with ip injections, once a day, of sterile saline or 20 μg / mouse of swainsonine hydrochloride in sterile saline. Tumor size is measured twice a week with calibrators and tumor weights are measured 4 weeks after injection of tumor cells as is the method of Dennis, JW (Cancer Res. 50: 1867-1872, 1990) .

D. Determination of synergy of swainsonin hydrochloride with the interferon-inducing agent poly (IC) for inhibition of growth of solid tumors The mice are provided with water to drink either with or without swainsonin hydrochloride (3.0 μg / ml) 2 days before they injected 105 MDAY-D2 tumor cells. Tumor diameters are measured with calibrators twice a week, and then on day 15 after injection of the tumor cells, the tumors are excised and weighed. The rate of tumor growth and the weight of the tumors at day 15 in the mice given swainsonin hydrochloride as a supplement in the drinking water and / or two i.p injections. of poly (I.C.) are compared as described in Dennis JW Cancer Res. 46: 5131-5136, 1986.

E. Improvement of the antiproliferative effect of interferon in vitro by swainsonine hydrochloride Human carcinoma cells HT29m, SN12C11 or MeWo melanoma cells are seeded in 5% FBS in culture medium of MEM tissue at 103 / ml in the presence and absence of approximately swainsonine hydrochloride (1.2 μg / ml) with or without 1000 Ul / ml of human interferon alpha-2 (intronA, Schering-Plow). The cells are cultured at 37 ° C in an atmosphere of C02 5% and on day 5 the cell number is determined. The method is as described by Dennis, J.W. JNCI 81: 1028-1033, 1989.

F. Screening of progenitor cells in vitro At the specified times after treatment with between 0.7 and 5.0 μg / ml swainsonine hydrochloride, control and treated mice are sacrificed by cervical dislocation. The bone marrow (BM) and the spleen cells of each are processed according to the procedures of the GIBCO-BRL mouse bone marrow pluripotent cell proliferation kit (Cat. # 3827SA, Grand Island, NTY). The potential colonies that are formed in the semi-solid medium are CFU-GEMM, CFU-GM and BFU. The plates are incubated for 10-14 days at 37 ° C in a humidified atmosphere of 5% C02 and 95% air, and colonies consisting of at least 40 cells are enumerated using an inverted microscope (20X magnification) to demonstrate stimulation. of the growth of hematopoietic progenitor cells.

G. Bone marrow proliferation assay The mice are treated with either 3 μg / ml of swainsonin hydrochloride in the water to be drunk or injected with 20 μg / mouse of swainsonin hydrochloride daily for 2-6 days. Proliferation is determined by the incorporation of [3 H] -thymidine (5 μCi / ml) for 18 hours at 37 ° C in cultures containing equal amounts of freshly isolated BM cells in complete medium. The radiolabeled cells are harvested with the aid of a cell harvester on glass filters, and the radioactivity is determined using a liquid scintillation counter. The cellularity of the bone marrow is also determined using the Coulter counter by directly counting BM cells after they have been washed from the tibias and femurs.

H. In vivo progenitor assay: Spleen colony formation assay They are irradiated with x-rays to mice (10-14 weeks of age) for a total body exposure of 700cGY. Irradiated mice are maintained with sterile drinking water and approximately 3 μg / ml) and antibiotics are administered to minimize mortality from infection. The number of BM pluripotent cells is determined by the Till and McCulloch method, which is based on the ability of progenitor pluripotent cells injected intravenously to form colonies in the spleens of recipient mice previously exposed to a lethal dose of whole body irradiation. The number of CFU is proportional to the number of pluripotent hematopoietic stem cells present in the hematopoietic graft. Ten days after the transplant, the recipient mice are sacrificed, their spleens are removed and fixed in Bouin's solution and the colonies visible in a general manner are counted.

I. Transplantation and repopulation of bone marrow Before transplantation with bone marrow cells, mice are pretreated with either a lethal dose of a chemotherapeutic agent or a lethal dose of x-ray radiation, as described in White et al (Cancer Communictions 3:83, 1991) and Oredipe et al. (JNCI 83: 1149, 1991). Mice aged 10-14 weeks are irradiated using Phillips RT 250 x-ray machines (two opposed therapeutic x-ray machines of 250 kbp, 235 kb, 15 mA, filtration 0.25 copper and 0.55 aluminum, with a mean layer of 0.99). mm of copper). Irradiation occurs with a dose rate of 126 cGy / min (63 cGy / min X 2) for 5 minutes and 33 seconds, for a total full body exposure of 700 cGy. This level of exposure to irradiation is within the range described as fatal for mice. After irradiation with x-rays, the animals are infused with freshly prepared bone marrow cells from donor mice either controlled or treated with swainsonin hydrochloride. The donor mice treated with swainsonin hydrochloride receive approximately 20 μg / ml swainsonine hydrochloride for 6 days. The recipient mice are monitored for survival for a period of 30 to 50 days.

J. Thl immune response: natural killer cell (NK) and killer activated lymphokine (LAK) assays Human peripheral blood mononuclear cells (PBM) are isolated from whole blood using standard methods (Rees et al, J. Immunol Meths., 62: 79-85, 1983; or Sedman et al, Br. J. Surg. : 976-981, 1988). They are plated in six-well plates in 5 ml cultures at a concentration of 1.5 million cells per ml either alone (control) or with varying concentrations of swainsonin hydrochloride together with one thousand international units (Ul) / ml of IL-2 for 3 days for the LAK assay or 1000 IU / ml interferon-alpha overnight, for the NK assay. The NK cell activity of PBMCs cultured in a Cr51 release assay is measured using the K562 cell line (NK cell sensitive) as target cells. The activity of LAK cells is measured using a Daudi cell line labeled with Cr51 (resistant to NK cells) as targets.

K. Measurement of STAT Concentrations and Activation as a Thi / Th2 Immune Response Differentiation Medium To measure the level and activation of STATs, DBA / 2 mice are treated for 6-9 days with 20 mg / ratdn / day of swainsonin hydrochloride followed by a single intraperitoneal (ip) injection of either sterile saline or of 100 mg of poly IC (ie, dsRNA, one surrogate for virus) in sterile saline. Two hours after an optimal time for STAT activation, the vessels of the mice are homogenized and cytosolic and nuclear cell extracts are prepared. The levels of STAT protein in the cytosolic and nuclear fractions are measured by Western blot analysis. STAT phosphorylation (i.e., activation) is measured following immunoprecipitation using antibodies against phosphotyrosine. Mice treated with 20 mg / day ip of the swainsonin hydrochloride salt exhibit enhanced STATl cytosolic protein levels while STAT3 remains unchanged (see Figures 10B to 10C). The following is a detailed description of Fig. 10A to 10C: Fig. 10A illustrates SW hydrochloride increases activation of STATl in spleen following treatment of DBA / 2 mice with poly IC. DBA / 2 mice receive daily ip injections of SW hydrochloride (20 μg / day) for 10 days. On day 11, mice are injected with poly IC (100 μg / mouse) or an equivalent volume of PBS 2 h before being sacrificed. The spleen and liver tissues are collected and immediately frozen in liquid nitrogen. Nuclear extracts are prepared and analyzed (8 μg) by immunoblotting with the indicated antibodies. Similar results are observed in liver (data not shown). Figure 10B. Cytosol extracts are prepared and analyzed (8 μg) by immunoblotting with the indicated antibodies. Spleen nuclear extracts are prepared and analyzed (20 μg) by immunoblotting with antibodies against phosphotyrosine. Figure 10C. Activation of STAT and turnover of activated STAT which occurs rapidly in response to the IFN type I poly IC inducer. DBA / 2 mice receive a single ip injection with poly IC (100 μg / mouse) and are sacrificed at the indicated times . Alternatively, an ELISA or ELISA-like assay can be used to detect STAT levels and activation in human peripheral blood. STAT dimers, attached to DNA promoter consensus sequences which have been bound to plastic microtiter plates, are detected using antibodies against STAT coupled to alkaline phosphatase (or other appropriate label). The samples of human peripheral blood lymphocytes are lysed and the cell extracts are prepared by methods known in the art. The levels of bound activated STAT protein are quantified by an optical medium after reaction of bound STAT protein with an appropriate detector (for example, if antibodies coupled to alkaline phosphatase are used then a alkaline phosphatase reactive colorimetric substrate can be used for detection). ).

L. Activity in the mouse hepatitis model The activity of the drug against viral hepatitis can be determined by strains of mice infected with mouse hepatitis virus-3 (MHV-3). Previous studies with MHV-3 have focused on mouse strains which develop fulminant hepatitis (Balb / cJ) or show resistance (A / J) to MHV-3 (Yuwaraj et al., 1996). Strain CH3 / HeJ, which develops chronic hepatitis in response to MHV-3 infection, is treated with saline or swainsonin hydrochloride (20 μg / mouse / day) alone or in combination with IFN. Before and during the treatment, levels and STAT activation status (as described under "K") are measured, as well as serum cytosine levels, viral load and survival.

M. Activity in patients with chronic hepatitis C Response to treatment with swainsonine hydrochloride or swainsonine hydrochloride plus interferon-alpha in patients with chronic hepatitis C can be monitored by a decrease in viral load and serum alanine aminotransferase (ALT) measured during treatment, for example 3, 6 and 12 months. An improvement in liver histology can also be determined by performing biopsies before and after treatment. The swainsonine hydrochloride is administered orally, twice a day, the dose between 50 and 200 μg / kg, and either alone or in combination with interferon alfa, which is administered at doses of 1 to 3 MU three times weekly. During this time, swainsonin hydrochloride can be administered continuously or intermittently (for example 2 weeks if, one week not). The response in patients receiving swainsonin hydrochloride is compared with the response in patients receiving placebo or interferon alfa. The detection of hepatitis C viral RNA in serum, liver and peripheral blood mononuclear cells is carried out by the polymerase chain reaction and reverse transcriptase (RT-PCR) method, using a specific primer for the 5 'untranslated region. conserved (UTR) for qualitative detection or, with an appropriate internal control RNA, for quantitative detection. The second method is a signal amplification or a branched-chain DNA (bDNA) assay. The viral nucleic acids are hybridized in microtitre plates and reacted with elongation probes specific for the virus followed by DNA polymerases. For an improvement in liver histology, the index of histological activity is used based on a qualification system developed by Knodell et al (Hepatology 1981, 1: 431-435), which assigns degrees in four categories: periportal necrosis, interlobular necrosis , portal inflammation and fibrosis. Alternatively, a system based on liver inflammation score (0-4) and stages of fibrosis (0-4) can be used (Scheurer PJ, J. Hepatol 1991; 13: 372-374).

N. Hemorres aura / uimiopro ección Cell models of hemorrhage / chemoprotection animals are described in Oredipe et al, 1991, supra, and White et al, 1991, supra. Although the present invention has been described with reference to what is currently considered to be the preferred examples, it should be understood that the invention is not limited to the described examples. On the contrary, it is intended that the invention encompass various modifications and equivalent arrangements included within the spirit and scope of the appended claims. All publications, patents and patent applications are hereby incorporated by reference in their entirety to the same extent as are individual publications, patents or patent applications which are specifically and individually indicated, incorporated by reference in their entirety.

Table 1 Atomic coordinates (x 104) and equivalent isotropic displacement parameters (A2 x 103) for Swainsonin HCl. U (eq) is defined as one third of the trace of the orthogonalized Uij tensor Union lengths [A] and angles [degrees] for Swainsonin Hydrogen coordinates (x 104) and isotropic displacement parameters (A2 x 103) for Swainsonin.

Table 2 Atomic coordinates (x 104) and equivalent isotropic displacement parameters (A2 x 103) for Swainsonin HBr. U (eq) is defined as one third of the trace of the orthogonalized Uij tensor Union lengths [A] and angles [degrees] Hydrogen coordinates (x 104) and isotropic displacement parameters (A2 x 103).

Table 3 Galustian et al., Immunopharm, Mononuclear cells of 27: 165, 1994 human peripheral blood in culture with human erythroblastoid tumor cell lines, K562 (NK-sensitive target) and human colorectal CoLo 320 (LAK-sensitive target) Mohla et al., Anticancer Res. Human carcinoma cells 10: 1515, 1990 human MCF-7 (estrogen receptor negative) and MDA-MB-231 (estrogen receptor positive) injected in athymic nude mice Table 4 Stability of SW hydrochloride, SW free base and SW hydrobromide Table 5 Other physical properties of Swainsonin hydrochloride (SW = swainsonine) Table 6 BEST SQUARE PLANS Swainsonin hydrochloride (plane defined by? L C9, C8, C7) Deviations of the plane atom NI 0.052 Á C9 -0.079 Rms 0.066 C8 0.078 C7 -0.050 C6 '0.0671 Á out of the top plane Swainsonin diacetate Deviations of the plane atom NI -0.023 Á C9 0.034 Rms 0.029 C8 -0.034 C7 0.022 C6 0.644 Á out of the top plane Swainsonin Bromhydrate Deviations of the plane atom NI 0.042 Á C9 -0.064 Rms 0.053 C8 0.062 C7 -0.040 C6 0.0673? out of the top plane Table 7 Chemical shifts of 1H from SW and SWHCl samples in D20 a Protons 3 and 3 'correspond to the pseudo-equatorial and pseudo-axial positions, respectively, in the 5-membered ring. Estimated chemical shift, due to overlap with H-3 in SW and H-31 in SWHCl.

Table 8 Selected constants of 1H-XH coupling of SW and SWHCl samples in D20 Table 9 13C chemical shifts of SW and SWHCl samples in D, 0 Table 10 NMR summary table on carbon and APT band assignments Table 11 Summary table of quantitative microanalytical results of Swainsonin hydrochloride (1) Determined by combustion analysis (TP 10812). (2) Determined by potentiometric titration analysis (TP 10812). (3) Calculated by difference. (4) Determined by Karl Fischer calorimetric titration in Phoenix Labs. (5) Determined by upper space gas chromatography analysis (in all there were 16 organic solvents tested by Phoenix Labs), ND = Not Detected. (6) Determined by U.S.P. < 281 > , residue when subjected to ignition (TP 18038).

Table 12 Summary table of infrared band assignments Table 13 Summary of the spectral mass fragmentation scheme Table 14 Effect of SW or SWHCl on the growth of early erythroid colonies of 2 x 10 5 nucleated cells BM * The data is the average of UFC-E of accounts by + DE. ** p, different from the control in the Student's t-test

Claims (29)

1. A stable chloride or bromide salt of swainsonin.

2. The crystalline chloride salt of swainsonin, as described in claim 1, comprising molecules of salts of chloride of swainsonin that are held together by hydrogen bonding interactions.

3. The crystal bromide salt of swainsonin, as described in claim 1, comprising molecules of swainsonin bromide salts that are held together by hydrogen bonding interactions.

4. The chloride or bromide salt of swainsonine, as described in claim 1, comprising four molecules of chloride salts or of swainsonine bromide in a unit cell.

5. The chloride or bromide salt of swainsonine, as described in claim 1, comprising molecules of hydrochloride salts or swainsonin hydrobromide.

6. The crystalline hydrochloride salt of swainsonin, as described in claim 5, wherein the salt molecules of swainsonine hydrochloride are held together by hydrogen bonding interactions of nitrogen and oxygen atoms of a first molecule of a salt of swainsonine hydrochloride to chloride ions of other molecules of a swainsonine hydrochloride salt.

7. The crystalline hydrobromide salt of swainsonin, as described in claim 5, wherein the salt molecules of swainsonin hydrobromide are held together by hydrogen bonding interactions of the oxygen atoms of a first hydrobromide salt molecule. swainsonin to bromide ions of other molecules of a swainsonin hydrobromide salt, and an interaction of hydrogen bonds of the nitrogen atom of the first molecule to an oxygen atom of a second molecule of a swainsonin hydrobromide salt.

8. The crystalline hydrochloride or hydrobromide salt of swainsonin, as described in claim 1, which has a group symmetry in space P212121.

9. The crystalline hydrochloride or hydrobromide salt of swainsonin, as described in claim 5, which has a group symmetry in space P212121.

10. The crystalline hydrochloride or hydrobromide salt of swainsonin, as described in claim 9, wherein the unit cell is orthorhombic.

11. The crystalline hydrochloride salt of swainsonin, as described in claim 10, which has the unit cell lengths: a = 8.09 + 0.01 A, b = 9.39 + 0.01 A, and c - 13.621 + 0.01 A.

12. The crystalline hydrobromide salt of swainsonin, as described in claim 10, which has the unit cell lengths: a = 8.40 + 0.01 A, b = 8.63 + 0.01 A, and c = 14.12 + 0.01 A.

13. The crystalline hydrochloride salt of swainsonin, as described in claim 11, having the atomic coordinates shown in table 1.

14. The crystalline hydrobromide salt of swainsonin, as described in claim 11, having the atomic coordinates shown in table 2.

15. A composition comprising a stable chloride or bromide salt of swainsonin.

16. The composition as described in claim 15, wherein the chloride or bromide salt is a hydrochloride or hydrobromide salt.

17. A method for preparing a crystalline swainsonine hydrochloride salt, as described in claim 5, comprising treating swainsonin acetonide with hydrochloric acid, and purifying the halide salt by crystallization and without chromatography to produce a crystalline hydrochloride salt of swainsonin .

18. A method for stimulating the immune system, treating proliferative disorders or microbial or parasitic infections in a subject comprising administering to a subject an effective amount of a composition as described in claim 15.

19. A method for the treatment of cancer, characterized in that it comprises administering to a subject an effective amount of a composition as described in claim 15.

20. The method described in claim 19, wherein the treatment comprises inhibiting metastasis or neoplastic growth.

21. A method stimulating the growth of hematopoietic progenitor cells, comprising administering to a subject an effective amount of a composition as described in claim 15.

22. The method described in claim 21, wherein the patient has been administered a myelosuppressive agent or is a bone marrow transplant recipient.

23. A method treating viral, bacterial, fungal or parasitic infections in which elimination of the pathogen requires a Th 1 response in a subject, comprising administering to the subject an effective amount of a composition as described in claim 15.

24. A method treating hepatitis C, which comprises administering to a subject an effective amount of a composition ulated from swainsonin free base, a swainsonin halide salt, or combinations thereof.

25. A method increasing the immunogenicity of a vaccine, comprising administering a stable chloride or bromide salt of swainsonin, as described in claim 1.

26. A method using atomic coordinates of the chloride salt or purified crystalline bromide of swainsonin as described in claim 1 or portions thereof computationally evaluating a chemical entity inhibition of Golgi mannosidase II.

27. The use of a purified chloride or bromide salt of swainsonin, as described in claim 1, in the manufacture of a pharmaceutical composition stimulating the immune system, treating proliferative disorders or microbial or parasitic infections.

28. The use of a purified chloride or bromide salt of swainsonin, as described in claim 1, in the manufacture of a pharmaceutical composition the treatment of cancer.

29. The use of a purified chloride or bromide salt of swainsonin, as described in claim 1, in the manufacture of a vaccine.