Patent GB-817385-A (GB)
Title: Process for the production of an antibiotic designated kanamycin
IPC: C22F1/05
Filed: 1957-09-02
Abstract: <PICT:0817385/IV (b)/1> A new antibiotic, kanamycin, is produced by the aerobic cultivation of a kanamycin-producing strain of Streptomyces kanamyceticus nov. spec. e.g. S. kanamyceticus NTCC 12853, in an aqueous nutrient medium containing a source of carbon, nitrogen and inorganic salts.  Specified carbon sources are starch, dextrin, maltose, glucose, sucrose, lactose and glycerol. Specified nitrogen sources are nitrates, soybean meal, cottonseed meal, peanut meal, meat extract, peptone, corn steep liquor and yeast extract. Inorganic salts are magnesium sulphate, manganese chloride, zinc sulphate, sodium chloride, potassium chloride, potassium monohydrogen phosphate and calcium carbonate.  A preferred medium contains starch, glucose, soybean meal, sodium chloride, potassium chloride, magnesium sulphate, potassium monohydrogen phosphate, calcium carbonate, sodium nitrate, manganese sulphate and zinc sulphate.  The initial pH of the medium is 7.0, the temperature of cultivation 25 to 35 and especially 27 DEG  to 32 DEG  C. and the duration of fermentation 2 to 7 and especially 3 to 5 days.  Karamycin is isolated from the broth by (a) solvent extraction, or (b) adsorption.  In the case of (a) the (whole or clarified) broth is extracted with butanol in the presence of lauric acid or stearic acid at pH 6-8 and then with an aqueous medium at pH 2 to 4.  An extraction with butanol, ethyl acetate, butyl acetate or benzene may be made to remove impurities.  Alternatively, the broth is concentrated, acidified and extracted with water, methanol, ethanol or acetone. Adsorption (b) may be on (1) activated carbon at neutral or alkaline pH, followed by elution with dry or aqueous alcohol such as methanol or acidified aqueous acetone of pH 2.0; (2) a cation exchange resin preferably of the carboxylic acid type, e.g. Amberlite IRC-50 (Registered Trade Mark) at pH 6.0 to 9.0, followed by elution with aqueous hydrochloric acid, aqueous sulphuric acid, aqueous acidified organic solvents such as acetone or aqueous ammonia.  The eluate is concentrated and a non-solvent added to precipitate kanamycin or its salt.  After initial isolation crude kanamycin may be further purified by column chromatography using alumina (treated with sulphuric acid) or active carbon.  Kanamycin is effective against Gram-positive and Gram-negative bacteria and mycobacteria: it is a basic compound forming salts with acids and containing 43.17 per cent carbon, 6.95 per cent hydrogen, 9.82 per cent nitrogen and 40.06 per cent oxygen (by difference); it is soluble in water and substantially insoluble in n-butanol, ethyl acetate, butyl acetate, ether, chloroform and benzene; Rf value in 0.2 per cent p-toluene sulphonic acid-butanol saturated with water is 0.21 to 0.26; [a ]1D = 112 DEG  (C = 1 per cent water); exhibits no absorption in the ultra-violet from 220 to 400 mm ; in infra-red kanamycin sulphate shows characteristic absorption bands at the following wavelengths expressed in microns 2.85, 3.03, 3.20, 3.30, 3.43, 3.63, 3.70, 3.83, 4.70, 6.05, 6.17, 6.27, 6.60, 6.90, 7.15, 7.35, 7.52, 7.60, 7.95, 8.18, 8.76, 8.95, 9.08, 9.40, 9.70, 10.20, 10.48, 10.75, 11.13, 11.50, 11.90, 12.35, 12.80 and 13.15. The antibiotic is distinguished from neomycin B and C and neamine. Kanamycin produces an N-acetyl derivative.  Kanamycin may be recovered from the fermentation broth as the hydrochloride or sulphate, which may be converted to the reineckate by treatment with ammonium reineckate.  The free base may be recovered from an aqueous solution of kanamycin sulphate by adding barium hydroxide to remove the sulphate ion.  Kanamycin may be used medicinally alone or in admixture with other drugs (see Group VI).ALSO:A therapeutic preparation comprises the antibiotic kanamycin (see Group IV (b)) and a pharmaceutical carrier.  Optional drugs which may be incorporated in the composition are antihistamines, sulpha drugs, stimulants, local anesthetics, analgesics, laxatives, sedatives, vitamins, hormones, antifungal agents and antibiotics such as streptomycin, tetracyclines and penicillin.