Patent GB-876096-A (GB)
Title: A new antibiotic, grisonomycin, and process for its manufacture
Filed: 1958-11-03
Abstract: <PICT:0876096/IV (b)/1> A water-soluble antibiotic grisonomycin active against many microorganisms is produced by growing streptomyces griseus A 10073 (F.I.T. Basle), or a mutant thereof, under aerobic conditions in an aqueous nutrient solution containing a source of carbon, nitrogen and inorganic salts.  The cultivation may be surface or submerged, the temperature is 18 to 40 DEG C. and the duration 1 1/2 to 5 days.  Specified carbon sources are glucose, saccharose, lactose, mannitol, starch and glycerol. Nitrogen sources are aminoacids, peptides, proteins and their degradation products such as peptone, tryptone, meat extracts, distillation residues, cereal grain water solubles and meals, e.g. soyabean meal, ammonium salts and nitrates.  Inorganic salts are chlorides, carbonates and sulphates of alkali metals, alkaline earth metals, magnesium, iron, zinc and manganese.  Grisonomycin is isolated from the clarified broth by adsorption.  The mycelium may be washed with water or an aqueous organic solvent to remove adsorbed antibiotic.  Adsorption may be on activated carbon, fuller's earth, or strongly acid ion exchange resins such as "Dowex 50" (Registered Trade Mark) and colour adsorbing resins, for example "Asmit" (Registered Trade Mark).  In the case of carbon, elution is by means of a mixture of water and an organic solvent, e.g. water-methanol, butanol/methanol/water with or without the addition of an acid, e.g. hydrochloric, formic or acetic or a basic agent, e.g. ammonia or aliphatic amine.  The antibiotic then being recovered by removing the solvent or adding a non-solvent, e.g. chloroform.  Where the eluate contains an acid or a base, it is preferred to neutralise the acid or base or to remove it by treatment with a weak anion-exchanger, e.g. Amberlite 1R4B or a weak cation exchanger "Amberlite 1RC50" (Registered Trade Mark).  In the case of adsorption on the resin Asmit, elution is by means of acidified aqueous methanol.  Further purification is obtained by distribution between aqueous hydrochloric acid and phenolic chloroform solutions. The antibiotic is recovered from the phenol chloroform mixtures by (a) precipitation with acetone, ether or petroleum ether, (b) precipitation as in (a) in the presence of a filter aid followed by dissolving in water, or (c) extracting with water after adding a mixture of ether and petroleum ether.  The above methods may be used singly or in combination.  Impurities may be precipitated by treatment with (a) picric acid, (b) picrolonic acid, (c) ammonium reineckate or (d) helianthin.  Excess precipitant may be removed from the filtrate by treatment with an ion exchanger such as Amberlite 1RA400.  Further purification may be obtained by chromatography on cellulose or other adsorbents previously referred to.  Grisonomycin is a pale yellow powder, soluble in water, aqueous alcohol, phenol and mixtures of phenol and organic solvents, insoluble in lipoid solvents, e.g. ether; has characteristic Rf values, e.g. 0.92 in water saturated with methyl isobutyl ketone; in the ultraviolet it exhibits two maxima at lambda= 280 mm  <FORM:0876096/IV (b)/1> and lambda=425 mm  <FORM:0876096/IV (b)/2> ; it has the infra red spectrum shown in the figure.  It is distinguished from streptomycin, rhodomycetia, actidion streptocia, grisoeia and candicidia.ALSO:A pharmaceutical preparation suitable for enteral, parenteral or local administration comprises an organic or inorganic carrier and the antibiotic grisonomycin (Group IV (b)).  The preparations may take the form of tablets, dragees, powders, salves, creams, suppositories, solutions, suspensions or emulsions.  Optional additions are preserving, stabilizing and wetting agents and other drugs. Specified carriers are gelatine, lactose, starch, magnesium stearate, talc, vegetable oils, benzyl alcohols, gums, alkylene glycols, cholesterol and petroleum jelly.