53179-09-2

Pathomyci n,Byk-Essex,W. Germany,1976

antibacterial, binds to ribosomes

Tank fermentation of Micromonospora inyoensis - Germination stage 1: Under aseptic conditions, add a lyophilized culture (or cells obtained from a slant culture) of M. inyoensis to a 300 ml shake flask containing 100 ml of the following sterile medium: Beef extract 3 g Tryptone 5 g Yeast extract 5 g Dextrose 1 g Starch 24 g Calcium carbonate 2 g Tap water 1,000 ml Incubate the flask and its contents for 5 days at 35°C on a rotary shaker (280 rpm, 2'' stroke).Germination stage 2: Aseptically transfer 25 ml of the fermentation medium of Germination stage 1 to a 2-l shake flask containing 500 ml of the above described sterile germination medium. Incubate the flask and its contents for 3 days at 28{]C on a rotary shaker (280 rpm, 2'' stroke).
Fermentation stage: Aseptically transfer 500 ml of the medium obtained from Germination stage 2 to a 14-l fermentation tank containing 9.5 l of the following sterile medium: Dextrin 50 g Dextrose 5 g Soybean meal 35 g Calcium carbonate 7 g Cobalt chloride 10-6M Tap water 1,000 ml Antifoam (GE 60) 10 ml Prior to sterilizing the above described medium, adjust the pH to 8. Aerobically ferment for 66 to 90 hours while stirring at 250 rpm with air input at 4.5 l/l/min and 25 psi. The potency of the antibiotic produced at the end of this period reaches a peak of 150 to 225 mcm/ml and remains relatively constant. The pH of the fermentation medium changes slightly during the antibiotic production, varying in the range of 6.8 to 7.3.
Isolation of Antibiotic 66-40 - The whole broth is adjusted to pH 2 with 6N sulfuric acid. (For the purpose of this example, quantities are given in terms of 170 l of fermentation broth obtained by pooling acidified broth from 17 batches.) The acidified broth is stirred for about 15 minutes and then filtered. Wash the mycelium with water and combine the washings with the filtrate. Adjust the pH of the filtrate to 7 with 6N ammonium hydroxide.
To the neutralized filtrate, add sufficient oxalic acid to precipitate calcium and filter. Reneutralize the filtrate with ammonium hydroxide. Charge the filtrate onto a cationic exchange adsorption column containing 1,500 to 2,000 g of IRC-50 Amberlite in its ammonium form. Discard the eluate, wash the resin with water, and elute with 2N ammonium hydroxide. Collect 400 ml fractions and monitor by disc testing with S. aureus ATCC-6538P. Combine active fractions and evaporate to dryness under vacuum obtaining about 28 g of crude Antibiotic 6640 having an activity of about 500 mcm/g.
Purification of Antibiotic 66-40 - Dissolve 28 g of crude Antibiotic 6640 in 100 ml of distilled water and charge to an anion exchange adsorption column (Dowex 1 x 2) in the hydroxyl form. Slurry 2,000 g of the resin in water into a column 2,5" in diameter and 36" high. Elute the column with distilled water at a rate of about 23 ml/min collecting 100 ml fractions and monitor with a conductivity meter and by disc testing against Staphylococcus aureus.
The disc testing provides a gross separation of antibiotic-containing eluate fractions from those devoid of antibiotic. To insure that the fractions are properly combined, a portion of each fraction is paper chromatographed using the lower phase of a chloroform:methanol:17% ammonium hydroxide system (2:1:1). Each paper is sprayed with ninhydrin and the eluates containing like material are combined and lyophilized yielding about 5.7 g of Antibiotic 66-40 assaying about 900 mcm/mg.

Siseptin (Schering).

Antibiotic

Although Sisomycin Sulfate has been approved for human use in theUnited States, it has not been marketed in this country. Itsantibacterial potency and effectiveness against aminoglycoside-inactivating enzymes resemble those of gentamicin.Sisomicin also exhibits pharmacokinetics and pharmacologicalproperties similar to those of gentamicin.

Poison by intravenous,intramuscular, intraperitoneal, and subcutaneous routes. When heated to decomposition itemits very toxic fumes of SOx and NOx.