Classifications

• • 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

Definitions

• the present invention refers to im unostimulant glucans, to a process for their preparation and to pharmaceutical compositions containing them.
• Glucan is a polysaccharide occurring in nature in the cell wall of fungine microorganism, particularly of yeasts.
• Glucans from different sources are different one from the other and moreover different extraction processes and treatments to which said microorganisms are subjected, including cultural and maintenance conditions, yield different final products. These differences can be noticed both in the three-dimensional structure of the polysaccharide chain, or in the chemical bonds between glucopyranoside units of said chain, and in the biological activity of the glucans as well as in the presence of substances other than glucan in the crude product with consequently greater or lesser difficulties in the purification.
• Glucans from Saccharomyces cerevisiae or from Lentinus edodes both having a branched structure with predominance of ⁇ -l,3-glucopyranoside bonds, are known.
• glucans are particularly studied because of their antitumoral and antibacterial activity (Int. J. Cancer 24, 773-779 (1979); Int. J. Immunopharmacol. Vol. 7 No. 5, 747-751 (1985)). Furthermore, they exhibit an immunomodulator effect both in vivo and in vitro (Rev. icrobiol. Vol. 15, 87-96, 1987) and exert a radioprotective action (Methods and Findings Explt. Pharmacol. Vol. 8, No. 3, 151-155 (1986)).
• Glucans have been produced also starting from Candida albicans and the immunomodulator effect thereof has been studied (J. Gen. Microbiol. Vol. 134, 1265-74 (1988)).
• EP-A-0416343 (16.08.1990) discloses the preparation of parietal glucanic bodies consisting of at least 90% glucan and partly of chitin, by extraction from the strain of Candida albicans ATCC 20955.
• the process for the preparation of this product comprises the treatment of the cells in autoclave and subsequent repeated extractions with sodium hydroxide and acetic acid at high temperature.
• US patent No. 4992540 (12.02.1991) discloses glucans extracted from Saccharomyces cerevisiae as alimentary additives.
• the glucans of the invention have the following characteristics: ratio between ⁇ (l-3) and ⁇ (l-6) bonds equal to about 1:1; - chitin content from 3 to 5% by weight; residual protein content lower than 0.3%; absence of mannane; enhancing activity of the in vitro NK cytotoxic activity.
• the glucans of the invention are obtainable by different yeast species.
• Candida albicans ATCC 20955, disclosed in EP 0416343 ⁇ is preferred, the glucans of the inventions may be obtained from a number of different strains of Candida, Saccharomyces or other yeast or mycetes species.
• the extraction process of the glucans of the invention from the cells comprises the following steps: a) culture of the microorganism in liquid medium, with low glucose content; b) treatment of the cell mass in autoclave at temperatures higher than 100°C; c) repeated extractions with sodium hydroxide and diluted organic acid; d) treatment of the extract with detergent at high temperature.
• steps a)-c) are substantially similar to that described in EP0416343, the step d) has never been described and contributes to the peculiar characteristics of the glucans of the invention. These characteristics particularly comprise high immunostimulant activity, higher than that of known glucans, low toxicity and immunogenic activity.
• the treatment with detergent at high temperature is typically carried out using sodium 1-5% dodecyl sulfate, preferably about 2%, in a suitable buffer, from 1-3 hours at the boiling temperature.
• the step b) is preferably carried out at the temperature of about 120°C for 3 hours.
• the glucans are preparared starting from Candida albicans strain ATCC 20955.
• This strain univocally identified by means of the restriction polymorphism analysis of the cell DNA, has been deposited by the applicants on August 4, 1989 at the American Type Culture Collection according to the Budapest Treaty.
• the safest and most modern method to identify the biotype under exam is the cell DNA restriction polymorphism analysis (DNA restriction fragment lenght polymorphism; Magee et al. Mol. Cell. Biol. 8, 4721, 1988).
• the restriction pattern of the strain provides a genetic fingerprint of - ne microorganism and turns out to be different from th c. of all the other men:, ers of the Candida genus.
• Biochemical characteristics it ferments glucose and maltose with production of acid and gas, saccharose with the production of acid only and it does not ferment lactose.
• Bio characteristics it is pathogenic for rabbit and mouse.
• the Candida albicans strain ATCC 20955 is kept on Sabouraud agar Difco in refrigerator at 4°C after 24 h growth at 28°C.
• the yeast is grown on a medium having a low glucose content so as to favour the production of the cell-wall, e.g. Winge medium, containing glucose and yeast extract, at 28 ⁇ C for 18-24 hours, monitoring the culture and checking for the presence of the yeast phase only, so as to obtain an optimal glucose-chitin ratio of approximately 20:1.
• the cells grown in the culture broth are collected by centrifugation, washed three times with sterile distilled water and suspended again (1-2% w/v) in pH 5 citrate buffer and then placed in autoclave at 121°C for 3 hours so as to cause the rupture of the cells, the solubilization of the fraction consisting of mannan, proteins, mannoproteins and the release of most cell components.
• the mass is collected by centrifugation, resuspended (l%-2% w/v) in 1% sterile NaOH and heated to 100°C for 24 hours. The mass is then washed three times with sterile distilled water until neutral reaction and then resuspended (1-2% w/v) in sterile 0.5 M acetic acid and treated at 80°C for 24 hours after having being washed three times with sterile distilled water until neutral reaction.
• the obtained glucan is further purified by treatment (1-2% w/v suspension) with a 2% sodium dodecylsulfate solution in Tris EDTA mercaptoethanol for 1,5 hours at the boiling temperature.
• the product is washed by centrifugation with sterile distilled water until all the detergent is removed.
• the obtained glucan may also be sterilized in autoclave at 121 ⁇ C for 30 minutes and finally it is freeze-dried.
• the obtained product is insoluble in water, methanol, acetone, ethyl ether, diluted acids and alkali, partially soluble in warm 1 M NaOH (0,06%) and soluble in dimethylsulfoxide; it contains 95-97% glucan together with 3-5% of chitin with a protein content lower than 0.3% (usually from 0.1 to 0.3%) and
• glucans are not antigenic and exhibit biological activities which classify them as Biological Response Modifiers.
• studies carried out on mice showed that the administration of the glucans can induce an increase of the anti- infective activity induced by polymorphonucleates leukocytes and activated macrophages both against chronic and acute infection; they also enhance the antitumor activity due to NK cell and activated macrophages; they significantly increase the interleukin production and particularly that of tumor necrosis factor Ot and interleukin 2; they potentiate the antibody response.
• the immunoadjuvant activity in the animal by the parenteral route is very high without remarkable side- effects being noticed and also by the oral route the activity is very interesting, above all as far as the activity on the lung alveolar macrophages is concerned, also perfectly tolerated.
• the high purity of the glucans of the invention imparts to the molecule particularly interesting characteristics from the point of view of tolerability: acute and chronic toxicity tests did not show any toxic local or systemic effect ( D 5Q > 1000 mg/kg i.p. in the mouse and in the rat an LD 5Q >2000 mg/kg p.o. in the mouse and in the rat, no toxic effect after daily administrations repeated for one year with doses up to 400 mg/kg/die or up to 250 mg/kg/die i.p.). Moreover no mutagenic, teratogenic, embryotoxic properties or anyhow influencing fertility have been noticed.
• a loopful of C. albicans agar is inoculated in 100 ml of Winge broth (Difco glucose 0.3%, 0.1% Difco yeast extract in distilled water, pH 6.5). The organism was grown at 28°C, under slight stirring (50 rpm) for 18-24 hours until the stationary growth phase was reached (about 2.8 x 10° cells/ml, corresponding to approx. 14 mg of dry weight/ml).
• 100 ml of broth culture is used to inoculate 1000 ml of Winge medium that are incub ⁇ t.ed as mentioned above.
• 1000 ml c broth culture previously obtained are used to inoculate 10 1 of Winge medium contained in the fermenter.
• the yeast was grown at 28°C, slightly stirred to 50 rpm, with a stream of air of 1 1/min. and the pH set on 6.5, until the stationary phase of growth Q was reached (about 2.8-4.5 x 10 cells/ml in 24 h). Control were performed during the growth to verify the presence of yeast cells only.
• the cells grown in the broth culture are harvested by low speed centrifugation (3000 rpm, 30 min), washed three times with distilled sterile water and re ⁇ suspended in citrate pH 5 buffer (223 g of citrate sodium/1 of distilled water) at a concentration of 2-4% and the suspension is autoclaved for 3 hours at 121 C C.
• the mass is harvested for centrifugation, re ⁇ suspended in 1% sterile NaOH at a concentration of about 2-4% and treated for 24 hours in an oil bath at 100 ⁇ C.
• the mass is then washed three times via centrifugation with sterile distilled water (neutral reaction) and it is harvested by centrifugation (5000 rpm, 30 min), re-suspended in sterile 0.5 M acetic acid at a concentration of about 2-4% and treated for 24 hours in an oil bath at 80°C.
• the mass is washed again three times by centrifugation with sterile distilled water (neutral reaction) .
• the mass is harvested by centrifugation, re- suspended at a ratio of about 2-4% in a 2% solution of sodium dodecylsulphate in Tris-EDTA-mercaptoethanol buffer (Tris - 0.1 M, EDTA 5 mM, mercaptoethanol 100 mM, pH 6.8) and boiled for 1.5 hours.
• Tris-EDTA-mercaptoethanol buffer Tris - 0.1 M, EDTA 5 mM, mercaptoethanol 100 mM, pH 6.8
• glucan is suspended in 25 ml of DMSO and stirred at 77°C until dissolution.
• the solution obtained is slightly stirred for 15' thereby adding 65 ml of distilled H-O. Addition of water provokes precipitation of glucan.
• the mixture obtained is slightly stirred for 5', after which other 6: ml of distilled water are added. The mixture is then centrifuged for 5' at 3500 rpm, and the supernatant discarded.
• the product collected for centrifugation after the last washing is transferred on trays and placed in an oven for 24 hours at 60 ⁇ C.
• the process yield is approx. 1.8-2.2 g of glucan per litre of initial culture broth.
• the 13C-NMR spectrum of the solubilized product has been recorded in DMSO-d 6 with a Bruker AC 300 apparatus at 75 MHz and 70 ⁇ C.
• the protein content according to Lowry is about 0.16% (0.78% in the glucan described in US 4992540).
• NK Activity (LU 10) of the peritoneal exudate of mice treated with the glucan of the invention (glucan from C. albicans) or with that of the US Patent 4992540 (glucan from S. cerevisiae)
• NK Activity (LU 10) of spleen cells of mice treated with the glucan of the invention (glucan from C. albicans) or with that of the US Patent 4992540 (glucan from S. cerevisiae)
• the glucans of the invention may be used for the treatment of tumoral diseases, bacterial or viral infections or of any condition in which a modulation of the immune system is desired.
• the glucans will be administered in form of pharmaceutical compositions suited to the oral, parenteral, rectal or topical administration.
• these formulations comprise tablets, capsules, sachets, syrups, solutions, vials, creams, gels, sprays and the like.
• the daily dosage will be determined by physicians according to the pathologies to be treated and to the patient's condition (weight, sex, age). It will be usually comprised between 0.1 and 50 mg/kg/die in one or more administrations.

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• 1992
  • 1992-08-10 IT ITMI921967A patent/IT1256035B/it active IP Right Grant
• 1993
  • 1993-08-03 AU AU47063/93A patent/AU4706393A/en not_active Abandoned
  • 1993-08-03 WO PCT/EP1993/002063 patent/WO1994003500A1/en not_active Application Discontinuation
  • 1993-08-03 EP EP93917731A patent/EP0654047A1/de not_active Withdrawn
  • 1993-08-06 MX MX9304793A patent/MX9304793A/es unknown
  • 1993-08-06 ZA ZA935730A patent/ZA935730B/xx unknown
  • 1993-08-06 CN CN93109362A patent/CN1082056A/zh active Pending

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