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WO1992013896A1 - Glucanes solubles - Google Patents

Glucanes solubles Download PDF

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Publication number
WO1992013896A1
WO1992013896A1 PCT/US1992/000866 US9200866W WO9213896A1 WO 1992013896 A1 WO1992013896 A1 WO 1992013896A1 US 9200866 W US9200866 W US 9200866W WO 9213896 A1 WO9213896 A1 WO 9213896A1
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Prior art keywords
glucan
sulfate
soluble
particulate
soluble glucan
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Inventor
David L. Williams
Rose Mcnamee
Nicholas R. +Di Di Luzio
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Bioglucans LP
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Bioglucans LP
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to a new class of soluble glucans and, more particularly, to soluble glucans in which the polyglucopyranose chains are solubilized by the addition of a polar charged chemical group as well as to processes for preparing
  • glucan refers generically to a 5 variety of naturally occurring homopolysaccharides or polyglucoses, including polymers such as cellulose, amylose, glycogen, laminarians, starch, etc.
  • Glucan encompasses branched and unbranched chains of glucose units linked by 1-3, 1-4, and 1-6 glucosidic bonds 0 that may be of either the alpha or beta type.
  • Particulate glucan designates a water-insoluble particulate (about 1-3 ⁇ ) polyglucose such as that derived from the cell wall of the yeast Saccharomyces cerevisiae.
  • Particulate 5 glucan is macromolecular and comprises a closed chain of glucopyranose units united by a series of 0-1-3
  • glucan phosphate or "soluble phosphorylated glucan” refers to the class of glucans solubilized by the addition of charged phosphate groups through reaction with phosphoric acid. These are the same or substantially similar to those substances as described in U.S. Patents Nos. 4,739,046; 4,761,402; 4,818,752 and 4,833,131.
  • Particulate glucan is a potent activator of the macrophage/monocyte cell series, complement, as well as T and B cell lymphocytes. Thus, particulate glucan has profound effects on both the reticuloendothelial and immune systems.
  • glucan Stimulation of the reticuloendothelial system by in vivo administration of particulate glucan leads to inhibition of allogenic or xenogeneic bone marrow graft acceptance in lethally irradiated animals.
  • This finding denotes that glucan will induce host defense mechanisms even against normal cells if they are genetically different from the host.
  • particulate glucan In addition to effects on reticuloendothelial and immune responses, in vivo administration of particulate glucan has been demonstrated to enhance hemopoietic activity including granulopoiesis, mono ⁇ ytopoiesis and erythropoiesis leading to greater recovery from a lethal dose of whole body irradiation (Patchen, 1983, Surv. Immunol. Res. 2.: 237-242) .
  • SUBSTITUTE SHEET when animals are challenged by microorganisms such as Eshericheria coli. Staphylo ⁇ occus aureus, Francisella tularensis. Mycobacterium leprae. Streptococcus pneumoniae. Candida albicans. Sporotrichum schenckii. as well as viruses such as Venezuelan equine encephalomyelitis virus, Rift Valley fever virus, murine hepatitis virus, frog virus III, Herpes simplex I and II, and parasites such as Leishmania donovani (see review by Di Luzio, 1983, Trends in Pharmacol. Sci. 4.: 344-347 and references cited therein) .
  • particulate glucan has potent anti-tumor activity.
  • particulate glucan has been shown to inhibit tumor growth and prolong survival in four syngeneic murine tumor models including adenocarcinoma BW 10232, anaplastic carcinoma 15091A, melanoma B16, and spontaneous lymphocytic leukemia BW5147 (Di Luzio et al, 1979, in Advances in Experimental Medicine and Biology, Vol. 121A: 269- 290) .
  • glucan preparation due to the particulate nature of the glucan preparation (1-3 ⁇ ) , it is difficult to administer via an intravenous route.
  • one patient receiving particulate glucan 5 required constant supervision during intravenous (IV) administration, continuous shaking of the IV drip bottle being essential to maintain the particulate glucan in suspension to avoid formation of emboli in the patient.
  • IV intravenous
  • slightly soluble neutral glucans are commercially available, these preparations are not suitable for intravenous administration because the aqueous solutions have very high viscosity and, more importantly, because their use when administered to experimental animals has inevitably been accompanied by considerable toxicity.
  • Lentinan a high molecular weight and poorly soluble j8-l,3 and 0-1,6 glucan obtained from Lentinus edodes. has been studied following intravenous administration to dogs. A variety of adverse clinical effects were observed following administration of lentinan (Ajinomoto Co. Inc., Tokyo, Japan) at doses of 2.0, 8.0 and 30 mg/kg/day for 5 weeks. Adverse effects included vomiting, erythema, discoloration of the sclera, and facial swelling. Circulatory collapse, unsteady gait, altered behavioral patterns. excessive salivation were also seen in individual beagles. At autopsy, congestion of the gastrointestinal mucosa was observed in animals treated with 2.0 or 8.0 mg/kg/day.
  • liver cells Morphological changes of liver indicated intracytoplasmic material, possibly lentinan, accumulating in liver cells.
  • One animal showed circulatory collapse upon the first injection at 8.0 mg/kg. While he did recover, the animal experienced repeated vomiting episodes with presence of blood indicating hemorrhaging of the gastrointestinal tract. Another animal appeared to show a marked allergic response, as demonstrated by erythema and subcutaneous swelling (edema) of the face.
  • Autopsy findings demonstrated extensive edema of subcutaneous tissue, and congestion of the gastrointestinal tract with hemorrhaging. Macrophage cells showed accumulation of material, possibly lentinan. (Chesterman et al.,1981, Toxicol. Lett. 9.: 87-90)
  • DMSO dimethylsulfoxide
  • particulate glucan dissolves in the presence of DMSO. All attempts to isolate a soluble glucan from the DMSO solution, however, resulted in failure.
  • a soluble glucan Upon dilution of the DMSO-glucan solution with various aqueous media such as glucose or saline solutions, the particulate glucan was reformed. Following dilution of the DMSO-soluble glucan solution with saline, all animals receiving injections of these solutions died immediately upon injection due to high concentration of DMSO or the reformation of the particulate glucan.
  • ethanol 100%
  • the precipitate was collected and lyophilized. When this lyophilized glucan was added to water, the particulate glucan reformed.
  • soluble phosphorylated glucan 0 (hereinafter termed "glucan phosphate”) through phosphoric acid hydrolysis using the method described briefly below.
  • glucan phosphate a stable solubilized form termed "soluble phosphorylated glucan" 0 (hereinafter termed "glucan phosphate”) through phosphoric acid hydrolysis using the method described briefly below.
  • This soluble phosphorylated glucan is non-toxic, non-immunogenic, and substantially non- pyrogenic (see U.S. Patent Nos. 4,739,046; 4,761,402; 5 4,818,752 and 4,833,131).
  • glucan phosphate was prepared as follows: particulate glucan was suspended in DSMO. Urea was added, the mixture heated and maintained at 50-150°C while phosphoric acid was o slowly added. The product was isolated and the DMSO, urea, glucose, and any unreacted phosphoric acid was removed.
  • the solubility of glucan phosphate as obtained from S. cerevisiae prepared according to the 5 above method is greater than about 50 mg/ml in water. Its elemental composition is illustrated in Table 1. (see U.S. Patent Nos. 4,739,046; 4,761,402; 4,818,752 and 4,833,131.)
  • the repeat unit empirical formula of glucan phosphate using this preparation is: 0 C 0 H 87 PO 37 .
  • glucan phosphate Therapeutic and prophylactic applications of glucan phosphate have been demonstrated to increase the host resistance of both normal and immunosuppressed animals to a variety of infectious diseases induced by bacterial, fungal, viral and parasitic organisms.
  • Glucan phosphate was also found to be useful in the treatment of neoplastic diseases, either alone or in combination with an anti- tumor agent. Studies have indicated that glucan phosphate stimulates macrophages to produce potent anti-tumor cytotoxins (see U.S. Patent No. 4,818,752). In addition, studies have demonstrated that glucan phosphate is useful in preventing the development of leukopenia induced by the administration of anti-cancer agents. (see U.S. Patent No. 4,739,046). Due to its ability to stimulate macrophage secretory and phagocytic activity, glucan phosphate has also been found effective in promoting wound healing. (see U.S. Patent No. 4,833,131).
  • the present invention provides a new class of soluble glucans (a) in which the polyglucopyranose chains have acquired a charged group from a non-phosphorous containing hydrolytic acid; (b) which are non-toxic; and (c) which surprisingly are capable of exerting pronounced immunobiological responses when administered in vivo in animals and humans.
  • These new soluble glucans immunostimulate macrophage activity with resulting activation of other immunoactive cells in the reticuloendothelial and immune systems. Additionally these soluble glucans enhance hematopoietic bone marrow activity.
  • These soluble glucans exhibit cytostatic effects against malignant neoplasms including melanomas and sarcomas in vivo.
  • the invention provides a process for producing a soluble glucan by solubilizing a particulate glucan, preferably prepared from Saccharomyces cerevisiae although other microbial sources may be used in a highly polar solvent which contains a strong chaotropic agent, reacting the resultant glucan with:
  • the invention provides a process for producing a soluble glucan by solubilizing a particulate glucan, preferably from Saccharomyces cerevisiae. although other microbial sources may be used, in a highly polar solvent which contains a strong chaotropic agent and reacting the resultant glucan with:
  • the invention provides methods for therapeutic and prophylactic treatment of infections induced by bacteria, fungi, viruses and parasitic organisms by administration of soluble glucans or pharmaceutical compositions comprising soluble glucans in combination with a physiologically acceptable carrier.
  • the present invention provides methods for treatment of malignant neoplastic disease in animals and humans, including but not limited to melanoma and reticulum cell sarcoma, by administering to an animal or a human a therapeutically effective amount of a soluble glucan alone or in combination with an anti-cancer agent. Furthermore, the invention provides methods for stimulating animal and human macrophage phagocytic activity by the administration of a soluble glucan. Methods are also provided for the stimulation of hematopoietic bone marrow activity by the 5 administration of a soluble glucan.
  • the immunobiological properties of the soluble glucans of the invention include (1) the ability to significantly modify viral infections; (2) the ability to modify infections induced by bacterial, 0 fungal and other parasitic microorganisms; (3) the ability to increase survival in animals and humans with malignant neoplasms; (4) the ability to enhance hematopoiesis as assessed by bone marrow proliferation; and (5) the ability to increase 5 macrophage phagocytic activity.
  • the soluble glucans are particularly useful for prophylactic and therapeutic applications against a variety of diseases induced by bacteria, viruses, o fungi and parasitic organisms, as well as a number of neoplastic conditions.
  • the soluble glucan compositions may advantageously be used with a physiologically acceptable pharmaceutical carrier, either alone or in combination with other bioactive or 5 pharmacological agents and therapeutic modalities.
  • FIG. l(A-C) illustrates the high performance size exclusion chromatograms for glucan sulfate A, 5 glucan sulfate B and glucan phosphate using an on-line multi-angle laser light scattering photometer.
  • FIG. IA is the chromatogram for glucan sulfate A. Two polymer peaks with molecular weight averages of 781,768.8 daltons and 5,710.4 daltons were resolved. 5
  • FIG. IB is the chromatogram for glucan sulfate B, Two polymer peaks with MW averages of 1,246,441 daltons and 14,513 daltons were resolved.
  • FIG. 1C included for comparison, is the chromatogram for glucan phosphate.
  • FIG. 2(A-B) illustrates helical coil transition analyses.
  • Dextran (70kD) served as the linear control and Congo red in sodium hydroxide served as the negative control.
  • FIG 2A is the helical 5 coil analysis for glucan sulfate A. It shows a shift in absorption maxima which is not consistent with that observed for compounds with a triple helical conformation.
  • FIG. 2B is the helical coil transition analysis of glucan sulfate B. It shows a shift in o absorption maxima which is characteristic of compounds exhibiting a triple helical conformation. Included for comparison in both FIG. 2(A and B) , is the helical coil transition analysis for glucan phosphate which shows the same shift characteristic of triple helical compounds. It served as the triple helical control for FIG. 2A and FIG. 2B.
  • FIG. 3(A-D) includes representations of the nuclear magnetic resonance spectrum for glucan phosphate, laminarin, glucan sulfate B and glucan sulfate A.
  • FIG. 3A included for comparison, is the 13 C-NMR representation for glucan phosphate at a concentration of 50 mg/ml.
  • FIG. 3B is the 13 C-NMR spectrum of a commercially available preparation of laminarin standard (Lot 95284 - K & K Laboratories, Inc. , Plainview, NY) at a concentration of about 30 mg/ml.
  • FIG. 3C is the 13 C-NMR representation for glucan sulfate B at a concentration of 50 mg/ml.
  • FIG. 3A included for comparison, is the 13 C-NMR representation for glucan phosphate at a concentration of 50 mg/ml.
  • FIG. 3B is the 13 C-NMR spectrum of a commercially available preparation of laminarin standard (Lot 9
  • FIG. 4 is a graph illustrating the effect of administration of a glucan sulfate on survival of mice with subsequent experimentally induced Candida albicans infection.
  • FIG. 5 is a graph illustrating the effect of prior treatment with a glucan sulfate on survival of mice with subsequent experimentally induced viral hepatitis.
  • Aqueous soluble glucans are prepared by a process comprising reacting a particulate glucan with a non-phosphorous containing hydrolytic acid containing a polar charged group.
  • useful, non-phosphorous containing hydrolytic acids include nitric acid, sulfuric acid, acetic acid, etc.
  • the polar charged group examples of which include nitrate, sulfate and acetate groups, is added onto the structure of the glucan in the presence of a highly polar solvent and a strong chaotropic agent.
  • soluble glucan sulfate is prepared by one of two processes, both which result in a unique class of products different from any other glucans previously described.
  • Soluble glucan sulfate prepared by the first method is herein designated as glucan sulfate A, while that prepared by the second method is herein designated glucan sulfate B.
  • glucan sulfate A is herein designated as glucan sulfate A
  • glucan sulfate B that prepared by the second method.
  • soluble glucan sulfate is prepared as follows: particulate glucan, derived from Saccharomyces cerevisiae. is suspended in a solution of a strong chaotropic agent in an aprotic solvent such as di ethylsulfoxide (DMSO) with constant stirring.
  • a strong chaotropic agent "relaxes” hydrogen bonding along the polyglucose chain, thus unfolding the molecule. It is preferred to use a fairly high concentration of a strong chaotropic agent such as urea ranging from about 4-12 M to prevent reformation of hydrogen bonds.
  • the mixture is then heated and maintained at about 50-150°C and sulfuric acid is added. A precipitate is apparent after about 1.5 hours.
  • the bioactive glucan sulfate product is isolated from the reaction mixture by resuspending or dissolving the precipitate by the addition of a sufficient amount of water and filtering the mixture through a coarse sintered filter.
  • the glucan sulfate is further isolated from the solution as described below.
  • soluble glucan sulfate is prepared by a preparation procedure which is substantially similar to the first method, with the following exception: a mixture of sulfuric acid (1/4 the amount employed in the first method) and DMSO is added to solubilize the particulate glucan, as opposed to sulfuric acid alone.
  • the DMSO is thought to buffer the hydrolytic effect of sulfuric acid, thus decreasing the degree of hydrolysis of the glucan polymers and increasing the percentage of conversion to the soluble material. This increases the yield from 37.5% using the first method to about 98% using the second method.
  • bioactive glucan sulfate product is isolated from the reaction mixture as follows: the mixture is cooled to stop the sulfation reaction and diluted with a volume of water sufficient to resuspend any precipitate. The resulting solution is filtered through a series of filters (e.g., l-3 ⁇ , 0.6 ⁇ , and
  • the solution is then molecularly sieved to remove all components of less than about 10,000 daltons molecular weight (MW) .
  • MW molecular weight
  • DMSO, urea, glucose and any unreacted sulfuric acid are removed from the solution.
  • Molecular sieving may be accomplished by any method that removes these low (i.e., less than about 10,000 daltons) MW components.
  • the solution is sieved using a Millipore dialyzer/concentrator with a 10,000 daltons MW membrane filter and a large volume of dialyzing solution. Following molecular sieving, the resulting solution is concentrated, shell frozen and lyophilized to yield the final product.
  • soluble glucan acetate is prepared as follows: particulate glucan, preferably derived from Saccharomyces cerevisiae. is suspended in a solution of a strong chaotropic agent, such as urea, in an aprotic solvent such as DMSO with constant stirring. It is preferred to use about 4-12 M of the chaotropic agent. The mixture is heated to about 50-100 ⁇ C and glacial acetic acid is added. No precipitate forms and the reaction is allowed to proceed for about 6 hours at about 100°C.
  • a strong chaotropic agent such as urea
  • DMSO aprotic solvent
  • particulate glucan is suspended in DMSO containing urea and the mixture is heated and maintained at about 50-150°C. A DMSO/acetic acid mixture is then added. This procedure does not result in the formation of a precipitate and the reaction is allowed to proceed for about 6 hours at 100°C.
  • the mixture is cooled to stop the reaction, diluted with deionized water, and filtered to remove any remaining particulate or colloidal material.
  • the solution is then dial zed and concentrated using, for example, tangential flow ultrafiltration. The resulting material is shell frozen and lyophilized.
  • the particulate glucan used in the processes for preparing the soluble glucans according to the present invention may be isolated from the cell wall of S_. cerevisiae by known methods (see e.g. , Di Luzio et al. , 1979, Internat'l J. Cancer 24.: 773-779; Hassid et al. , 1941, J. Amer. Chem. Soc. 63.: 295-298 incorporated herein by reference) . Briefly, in practice the particulate glucan is prepared as follows: dry yeast is digested in aqueous sodium hydroxide solution and heated to about 100°C and maintained for about 4 hours, then cooled overnight. The supernatant is decanted and the procedure is repeated three times.
  • the residue is acidified using hydrochloric acid and maintained at 100°C for about 4 hours, then cooled overnight.
  • the supernatant is decanted and the acid digestion is repeated twice.
  • the residue is then washed repeatedly with distilled water and extracted with ethanol for at least 24 hours.
  • the reddish-brown supernatant is then aspirated and discarded.
  • the ethanol extraction is repeated until the supernatant is essentially colorless.
  • the ethanol is removed by repeatedly washing the residue with distilled water.
  • the particulate glucan is collected by centrifugation or filtration.
  • soluble glucans can be prepared from neutral polyglucose or polyglucose-protein products derived from a variety of other microbial sources.
  • a non-exhaustive list of such sources is presented in Table 2.
  • the solubility of soluble glucan as obtained from S. cerevisiae prepared according to the first method of the present invention using sulfuric acid as the hydrolytic acid has been determined to be greater then 400 mg/ml in water.
  • the solubility of soluble glucan prepared according to the second method of the present invention using sulfuric acid as the hydrolytic acid (herein designated glucan sulfate B) is 50 mg/ml in water. Additionally, glucan sulfate A hydrates faster than either glucan sulfate B or glucan phosphate.
  • Glucan Sulfate B (C 6 H, 0 O 5 ) g . 3S0 3 NH 4 + "4H 2 0
  • the data from the high performance size exclusion chromatography indicate that there are two polymer or molecular weight peaks in both glucan sulfate A and glucan sulfate B (see FIG. IA and FIG. IB) .
  • the chromatogram of glucan phosphate (FIG. 1C) shown here for comparison, also indicates two polymer peaks.
  • Shift in maximal absorption to shorter wavelength at elevated pH indicates disruption of intramolecular hydrogen bonding, with subsequent transition of the helical conformation to that of a random coil, preventing the polymer from complexing with Congo Red.
  • Inhibition of formation of the glucan-Congo Red complex results in alteration in absorption spectra of the dye in the visible wavelengths between 400 and 600 nm.
  • glucan sulfate A shows a shift in the absorption maxima which is not consistent with that observed for compounds which are triple helical in nature. Although applicant does not intend to be limited to this explanation, it is presently thought that the response of glucan sulfate A may suggest that the molecule is composed of both linear and triple-helical polymers.
  • the data for glucan sulfate B show a shift in absorption maxima which is consistent with that observed for compounds which are triple helical in nature.
  • the shift in absorption maxima indicates that at least a portion of the polymers exhibit an ordered conformation.
  • the data for glucan phosphate (see FIG. 2C) are also shown for comparison. It too shows a shift in absorption maxima which is consistent with that observed for triple helical compounds.
  • the 13 C-NMR spectrum obtained for glucan sulfate B (see FIG. 3C) is consistent with 0-1,3 interchain linkages.
  • the spectrum obtained for glucan sulfate A (see FIG. 3D) is not. Based on current knowledge, it was not possible at present to make definitive carbon assignments for glucan sulfate A.
  • soluble glucans Due to the potent activity of soluble glucans in stimulating the immune response and reticuloendothelial system, soluble glucans are advantageously useful in prophylactic and therapeutic applications against diseases induced by a variety of microorganisms. Because soluble glucans influence very fundamental host defense systems of the body regulating the number, functional activity and interaction of macrophages, T and B lymphocytes, leukocytes and natural killer cells as well as their humoral and secretory components, they possess the potential for non-specifically modifying an extensive array of infectious diseases.
  • the soluble glucans may be used either alone or in combination with known antimicrobial agents to prevent and/or treat diseases induced by gram positive bacteria including, but not limited to:
  • Staphylococcus aureus Streptococcus pneumoniae. Mycobacterium tuberculosis. Haemophilus influenzae. Diplococcus pneumoniae: gram negative bacteria including, but not limited to: Escherichia coli. Bacterium enteritis. Francisella tularensis: acid-fast bacteria including, but not limited to: Mycobacterium leprae: viruses including but not limited to: Hepatitis; Herpes simplex I and II; etc.; fungi including, but not limited to: Candida albicans; Sporotrichum schenkii and protozoa1 parasites including but not limited to: Leishmania donovani. Schistosoma mansoni. etc.
  • the soluble glucans may be used for the prevention and/or treatment of opportunistic infections in animals and man which are immunosuppressed either as a result of congenital or acquired immunodeficiency or as a side-effect of chemotherapeutic treatment.
  • Soluble glucans of the present invention can be used either alone or in combination with a broad range of antimicrobial agents effective against diseases induced by bacteria, fungi, viruses and parasitic organisms.
  • Table 5 of U.S. Patent No. 4,761,402 presents a non-exhaustive list of some of the antimirobial agents that may be used in combination with the present soluble glucans. Table 5 is incorporated herein by reference.
  • Soluble glucans demonstrate a number of characteristics which make them particularly advantageous for the treatment of infections including, but not limited to the following advantages:
  • Soluble glucans have a broad range of activity. They are effective against infections induced by fungi and viruses, and may be used effectively against infections induced by bacteria and parasitic organisms; (2) Soluble glucans may be used additively or synergistically in combination with bioactive agents conventionally used to treat infections including but not limited to aminoglycoside antibiotics, etc. ;
  • Soluble glucans do not induce the development of resistance in causative organisms because their effects are mediated by the host; (4) Soluble glucans have very low toxicity;
  • Soluble glucans enhance a variety of diverse aspects of cellular and humoral immune responses of the host.
  • Soluble glucans stimulate hematopoiesis as evidenced by increased bone marrow proliferation.
  • Soluble glucans may be used to prevent or reverse the development of immunosuppression in the host. 5.3.2 THERAPY OF NEOPLASMS Due to the stimulation of macrophage phagocytic and secretory activity and increased proliferation of macrophages caused by soluble glucans, soluble glucans may advantageously be used either alone or in combination with other modalities such as surgery and chemotherapy, to treat malignant neoplastic diseases including, but not limited to adenocarcinoma, reticulum cell sarcoma, etc.
  • soluble glucans directly increase the median survival time of organisms challenged with tumor cells including, but not limited to, syngeneic melanoma and syngeneic reticulum cell sarcoma, thus suggesting they decrease the rate of progression of such diseases.
  • the soluble glucans of the present invention ca n be administered for prophylactic and/or therapeutic applications by a number of routes, including but not limited to: orally; by injection including but not limited to intravenously, intraperitoneally, subcutaneously, intramuscularly, etc. ; by topical application to nasal and nasopharyngeal linings; and by inhalation via aerosolization and application to respiratory tract linings, etc.
  • the soluble glucans When administered to an animal or a human, the soluble glucans may be combined with water, an aqueous solution or any physiologically acceptable pharmaceutical carrier or vehicle.
  • SUBSTITUTE SHEET 6 PREPARATION OF SOLUBLE GLUCANS Particulate glucan was prepared from Saccharomyces cerevisiae according to the method of Di Luzio et aJ- (1979, Int'l J. Cancer 24.: 773-779). Briefly, using a 6 1 flask, 540 gm of dry yeast (Universal Foods Corp. , Milwaukee, WI) was suspended in 3 1 of 3% aqueous sodium hydroxide solution. The suspension was placed in boiling water bath for 4 hours, cooled overnight and the supernatant decanted. This procedure was repeated three times.
  • the residue was then acidified with 800 ml of concentrated hydrochloric acid plus 2 1 of 3% hydrochloric acid and placed in a boiling water bath for 4 hours. The suspension was allowed to stand overnight and the supernatant decanted. The residue was further digested with 3 1 of 3% hydrochloric acid at 100"C for 4 hours, cooled overnight and decanted. The 3% hydrochloric acid digestion was repeated twice. The residue was then washed three times with distilled water (20°C) and twice with distilled water at 100°C. One 1 of ethyl alcohol was added to the residue, mixed thoroughly and allowed to stand a minimum of 24 hours for maximum extraction. The dark reddish-brown alcohol supernatant was aspirated from the residue and discarded. The alcohol extraction procedure was repeated until the alcohol supernatant was essentially colorless. The alcohol was removed by washing the residue four times with hot water; the particulate glucan preparation was then collected by centrifugation, frozen and lyophilized.
  • Glucan sulfate was prepared according to the present invention by solubilization and sulfation of the particulate glucan as follows: 72 g of urea (8 M) was added to a flask containing 200 ml dimethylsulfoxide (DMSO) and stirred until dissolved. Four grams of particulate glucan were added and stirred until dissolved. The flask was heated to about 100°C and 32 ml of concentrated sulfuric acid (14M) was added. The mixture was maintained at 100°C for 6 hours by immersion in a boiling water bath. It is preferred to allow the reaction to proceed for about 6 hours.
  • DMSO dimethylsulfoxide
  • the resulting solution was then molecularly sieved in order to remove low molecular weight (MW) fractions including glucose, DMSO, urea and unreacted sulfuric acid.
  • MW molecular weight
  • molecular sieving was accomplished using a Millipore dialyzer/concentrator (Millipore Corp. , Bedford, MA) with a 10,000 MW membrane filter. Dialysis against 100 L Milli-Q grade water was used to remove low MW compounds.
  • glucan sulfate A is stable in a lyophilized state for at least 2 years and at least for 15 months in solution maintained at 20°.
  • Glucan sulfate was prepared according to a second method of the present invention by solubilization and sulfation of the particulate glucan as follows:
  • the mixture was cooled and diluted with 4 L water to resuspend the 1 5 precipitate.
  • the mixture was then filtered through a Millipore pre-filter (about 1-3 ⁇ ) to remove the precipitate.
  • the resulting solution was then molecularly sieved in order to remove low molecular weight (MW) 2o fractions including glucose, DMSO and urea. After molecular sieving, the product was concentrated, shell frozen and lyophilized.
  • MW molecular weight
  • Group 1 (Harlan/Sprague-Dawley, Houston, TX) were divided into two groups.
  • Group 1 designated as the control group, received intravenous injections of dextrose (5% w/v) .
  • Group 2 received intravenous injections of glucan sulfate A (500 mg/kg) on days 10, 8, 6, 4 and 2 prior to the induction of candidiasis and on days 3, 6, 9 and 12 following the induction of candidiasis.
  • mice survival of mice was monitored for 60 days following infection with the fungus. Results are illustrated in FIG. 4.
  • Glucan sulfate treated mice showed a 75% long-term survival rate. In contrast, control mice which received dextrose showed only 8% long-term survival. 15 No further mortality was observed in either group past day 60.
  • mice Twenty C57B1/6J mice (Jackson Laboratories, Bar Harbor, ME) were divided into two groups. Group 1, the control group, received intravenous injections.
  • Group 2 received intravenous injections of glucan sulfate A (50 mg/kg) on days 10, 8, 6, 4 and 2 prior to the induction of acute viral hepatitis by intraperitoneal injection of a 1:2.5 dilution of 16 complement fixing units of mouse
  • mice survival of mice was monitored for 14 days following infection with the virus. Results are illustrated in FIG. 5.
  • mice showed a 60% long-term survival rate. In contrast, control mice which received dextrose showed 100% mortality. No further mortality was observed in either group after day 14.
  • Phagocytic function was evaluated by measuring the rate of intravascular clearance of 125 I- labelled reticuloendothelial test lipid emulsion.
  • 125 I- labelled RE test lipid emulsion was administered intravenously (30 mg/mouse) and vascular clearance was assessed at 1, 3, 6 and 9 min. Results are presented in Table 6.
  • Glucan sulfate A resulted in a significant (p ⁇ 0.01) 42% increase in the intravascular clearance of RE test lipid emulsion, thus indicating that glucan o sulfate A will increase in vivo macrophage phagocytic activity.
  • mice Twenty-four male C57B1/6J mice (Jackson Laboratories, Bar Harbor, ME) (about 18 g) were intravenously injected with glucan sulfate A (250 mg/kg) . Twenty-four control mice received isovolumetric dextrose (5% w/v) . Bone marrow cells were harvested at 24 hours. In vitro bone marrow proliferation was assessed by the method of Williams et. al. (1987, Hepatology 7: 1296-1304).
  • mice were sacrificed by ether euthanasia.
  • Bone marrow cells were collected by aspiration of the femur with physiological saline (3 ml) using a 25 gauge needle. Bone marrow cells were centrifuged, washed (2X) and resuspended to 1 x 10 6 /ml in RPMI-1640 media (Irvine Scientific, Santa Ana, CA) . Cells were aliquoted into 96 well plates (1 x 10 5 / well) containing RPMI-1640 with a final volume of 300 ⁇ l. Bone marrow cells were incubated for 40 hours and pulse-labelled with 3 H-thymidine (1 ⁇ Ci/well) for 8 hours.
  • mice Twenty C57B1/6J mice (Jackson Laboratories, Bar Harbor, ME) were divided into 2 groups of 10 each. All mice received subcutaneous injection of 5.0 x 10 5 syngeneic melanoma B16 cells. Thereafter, Group 1, designated as the control group, received dextrose (5% w/v) twice weekly; Group 2 received glucan sulfate A (250 mg/kg) twice weekly. All injections were given intravenously or intraperitoneally. As demonstrated in Table 8, significantly increased median survival times (p ⁇ 0.001) were observed in mice treated with glucan sulfate A. However, ultimate survival (i.e., the time to 100% mortality) was not significantly altered in that population receiving glucan sulfate A.
  • mice were subcutaneously injected with 5.0 x 10 5 syngeneic reticulum cell sarcoma M5076 cells in the scruff of the neck on day 0. On day 0 and at 3 day intervals thereafter.
  • Group 1 consisting of 17 mice designated as the control group, received dextrose (5% w/v) intravenously;
  • Group 2 consisting of 12 mice, received glucan sulfate A (250 mg/kg) .

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Abstract

Nouvelle classe de glucanes solubles, ainsi que procédé de production desdits glucanes. Selon un mode d'exécution préféré, le glucane est un sulfate de glucane dérivé de la levure Saccharomyces cerevisiae. Les sulfates de glucane sont utiles pour les applications prophylactiques et thérapeutiques contre des infections induites par des bactéries, des virus, des champignons et des parasites. De plus, ils peuvent être administrés pour stimuler l'activité hématopoïétique de la moelle osseuse et pour stimuler par ex. l'activité phagocytaire des cellules macrophages in vivo. Lesdits sulfates de glucane sont également des agents utiles contre les maladies néoplasiques.
PCT/US1992/000866 1991-02-01 1992-01-31 Glucanes solubles Ceased WO1992013896A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5488040A (en) * 1989-09-08 1996-01-30 Alpha-Beta Technology, Inc. Use of neutral soluble glucan preparations to stimulate platelet production
US5622939A (en) * 1992-08-21 1997-04-22 Alpha-Beta Technology, Inc. Glucan preparation
US5622940A (en) * 1994-07-14 1997-04-22 Alpha-Beta Technology Inhibition of infection-stimulated oral tissue destruction by β(1,3)-glucan
US5633369A (en) * 1989-09-08 1997-05-27 Alpha-Beta Technology, Inc. Method for producing soluble glucans
US5786343A (en) * 1997-03-05 1998-07-28 Immudyne, Inc. Phagocytosis activator compositions and their use
US5849720A (en) * 1989-09-08 1998-12-15 Alpha-Beta Technology, Inc. Enhancement of non-specific immune defenses by administration of underivatized, aqueous soluble glucans
US6046323A (en) * 1997-07-29 2000-04-04 The Collaborative Group, Ltd. Conformations of PPG-glucan
US6117850A (en) * 1995-08-28 2000-09-12 The Collaborative Group, Ltd. Mobilization of peripheral blood precursor cells by β(1,3)-glucan
US6369216B1 (en) 1998-09-25 2002-04-09 Biopolymer Engineering Pharmaceutical, Inc. Very high molecular weight β-glucans
EP1206939A4 (fr) * 1999-07-21 2003-04-16 Yakult Honsha Kk Agents de reduction du cholesterol, inhibiteurs de production d'acide biliaire secondaire, aliments et boissons
US6632459B2 (en) 2000-12-11 2003-10-14 Nutricia N.V. Chlorogenic acid and an analog thereof for immune system stimulation
US7022685B2 (en) 1998-09-25 2006-04-04 Biopolymer Engineering, Inc. Very high molecular weight β-glucans
WO2015159134A1 (fr) 2014-04-14 2015-10-22 Uab "Biocentras" Composition thérapeutique de β-glucane, modulant le système immunitaire humain et initiant la rupture des cellules cancéreuses

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US4795745A (en) * 1984-09-19 1989-01-03 Larm Karl O P Macrophage-activating composition and a process for its manufacture
US4965347A (en) * 1986-03-03 1990-10-23 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Beta-D-glucan, and its production and uses

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US3985727A (en) * 1975-03-28 1976-10-12 Schering Corporation Aminoglycoside antibiotics
US4705780A (en) * 1979-01-25 1987-11-10 Univablot Medicaments containing pichia or extracts thereof
US4795745A (en) * 1984-09-19 1989-01-03 Larm Karl O P Macrophage-activating composition and a process for its manufacture
US4739046A (en) * 1985-08-19 1988-04-19 Luzio Nicholas R Di Soluble phosphorylated glucan
US4761402A (en) * 1985-08-19 1988-08-02 Bioglucans, L.P. Methods and compositions for prophylactic and therapeutic treatment of infections
US4965347A (en) * 1986-03-03 1990-10-23 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Beta-D-glucan, and its production and uses

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849720A (en) * 1989-09-08 1998-12-15 Alpha-Beta Technology, Inc. Enhancement of non-specific immune defenses by administration of underivatized, aqueous soluble glucans
US5532223A (en) * 1989-09-08 1996-07-02 Alpha-Beta Technology, Inc. Use of aqueous soluble glucan preparations to stimulate platelet production
US5633369A (en) * 1989-09-08 1997-05-27 Alpha-Beta Technology, Inc. Method for producing soluble glucans
US5663324A (en) * 1989-09-08 1997-09-02 Alpha-Beta Technology, Inc. Method for producing underivatized, aqueous soluble β(1-3) glucan
US5488040A (en) * 1989-09-08 1996-01-30 Alpha-Beta Technology, Inc. Use of neutral soluble glucan preparations to stimulate platelet production
US5811542A (en) * 1989-09-08 1998-09-22 Alpha-Beta Technology, Inc. Method for producing soluble glucans
US5622939A (en) * 1992-08-21 1997-04-22 Alpha-Beta Technology, Inc. Glucan preparation
US5783569A (en) * 1992-08-21 1998-07-21 Alpha-Beta Technology, Inc. Uses for underivatized, aqueous soluble β(1-3) glucan and compositions comprising same
US5817643A (en) * 1992-08-21 1998-10-06 Alpha-Beta Technology, Inc. Underivatized, aqueous soluable β(1-3) glucan, composition and method of making same
US5622940A (en) * 1994-07-14 1997-04-22 Alpha-Beta Technology Inhibition of infection-stimulated oral tissue destruction by β(1,3)-glucan
US6117850A (en) * 1995-08-28 2000-09-12 The Collaborative Group, Ltd. Mobilization of peripheral blood precursor cells by β(1,3)-glucan
US5786343A (en) * 1997-03-05 1998-07-28 Immudyne, Inc. Phagocytosis activator compositions and their use
US6046323A (en) * 1997-07-29 2000-04-04 The Collaborative Group, Ltd. Conformations of PPG-glucan
US7022685B2 (en) 1998-09-25 2006-04-04 Biopolymer Engineering, Inc. Very high molecular weight β-glucans
US6369216B1 (en) 1998-09-25 2002-04-09 Biopolymer Engineering Pharmaceutical, Inc. Very high molecular weight β-glucans
US7566704B2 (en) 1998-09-25 2009-07-28 Biopolymer Engineering, Inc. Very high molecular weight β-glucans
US7101544B1 (en) 1999-07-21 2006-09-05 Kabushiki Kaisha Yakult Honsha Cholesterol-lowering agents, secondary bile acid production inhibitors, and foods and drinks
KR100717742B1 (ko) * 1999-07-21 2007-05-11 가부시키가이샤 야쿠루트 혼샤 콜레스테롤 저하제，2차 담즙산 생산 억제제 및 음식품
US7413740B2 (en) 1999-07-21 2008-08-19 Kabushiki Kaisha Yakult Honsha Cholesterol-lowering agents, secondary bile acid production inhibitors and foods and drinks
EP1206939A4 (fr) * 1999-07-21 2003-04-16 Yakult Honsha Kk Agents de reduction du cholesterol, inhibiteurs de production d'acide biliaire secondaire, aliments et boissons
US7754204B2 (en) 1999-07-21 2010-07-13 Kabushiki Kaisha Yakult Honsha Cholesterol-lowering agents, secondary bile acid production inhibitors and foods and drinks
US6632459B2 (en) 2000-12-11 2003-10-14 Nutricia N.V. Chlorogenic acid and an analog thereof for immune system stimulation
US7288271B2 (en) 2000-12-11 2007-10-30 Nutricia N.V. Chlorogenic acid and an analog thereof for immune system stimulation
WO2015159134A1 (fr) 2014-04-14 2015-10-22 Uab "Biocentras" Composition thérapeutique de β-glucane, modulant le système immunitaire humain et initiant la rupture des cellules cancéreuses

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