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C (O VO r ca BY G NWLLWSNWL LNGOd teer a 7A/EIR A77OAAVAY June 18, 1957 F. W, TANNER, JR., ETAL 2,796,379 CARBOMYCIN A AND ITS PRODUCTION Filed Feb. 7, 1952 2. Sheets-Sheet 2 NRR 2325 Fig.2 S. halsted ii-Culture Med. Adjust o -1a pH3.o Mycelium Filter Neutralize off pH 6.5 Methyl Extract lso buity % vol. carbomycin Ketone from Aqueous Extract Twice Extract H2 SO4. Ketone carbomycin pH 2.0 off from Solvent H Wolsh Aqueous Benzene & Ketone Phose off NeutralizeA. pH e.s Extract carbo mycin Ether Dry over Na2SO4. Off Distill corbomycin

solid FRED W. T ANNER, Jr., THOMAS M. LEES, impur uttes JOHN 8. ROUT EN INVENTORS

BY C W saw-4. THER AT TORNEYS United States Patent Office E 1 2 m The complete cultural characteristics of the new strain, which is hereinafter designated NRRL-2331, are given in 2,796,379 Table I. (The colors where R is written are those of CARBOMYCINA AND ITS PRODUCTION 5 Ridgway, Color Standards and Nomenclature). Readings are based on six tubes except where otherwise noted. Fred W. Tanner, Jr., and Thomas M. Lees, Baldwin, N.Y., The new strain of S. haistedii, NRRL-2331, differs and John B. Rouien, Tenafly, N.J., assignors to Chas. from the description in Bergey's Manual in several re Pfizer & Co., Inc., Brooklyn, N.Y., a corporation of spects. These differences are shown in the following Delaware table. Applicationpp. Februaryary 7, 1952,SZ, serial No. 270,298 O TABLE 8 Claims. (C.167-65) Medium Strain NRRL-2331 Bergey's Description This invention is concerned with a new and useful anti 15 starch agar...... Moderate growth. White Abundant growth. biotic called Carbomycin A, and more particularly with EYSEE, E. Eysh, glossy its production by fermentation, methods for its recovery SE gray growtn. and concentration from crude solutions like fermenta- Glucose agar------Edgelichenoid,white, remaining Edgeoid, becomingbrown.of colony lichen tion broths, processes for its purification, and methods Potato plug------GE EEEEEE GEE. in for the preparationstarts rati of its salts. The invention includes 20 lium,with white no spores. aerial myce- klee,Growth cream-colored moist, wrin within its scope the antibiotic and its salts in dilute solu- with greetinge. tions, as crude concentrates and in pure crystalline forms. Synthetic agar.-- spiluEE though No spirals. These novel products are especially useful in vitro as disinfectants, and topically and internally as therapeutic agents in combatting pathogenic mycobacteria and various 28 It is to be understood that for the production of car gram-positive microorganisms. bomycin A the present invention is not limited to this The new antibiotic is formed during the cultivation, particular organism or to organisms fully answering the under controlled conditions, of a hitherto undescribed strain of a species of microorganism known as Strepto above description, which is given for illustrative purposes. myces halstedi. This new culture was planted and tested 30 In fact, it is especially desired and intended to include the on the media normally used for the identification of use of mutants produced from the described organism by actinomycetes and on the basis of the results secured, it agents such as X-radiation, ultraviolet radiation, nitrogen was identified as a new strain of Streptomyces halstedii, mustards, and the like. using the description of the actinomycetes given in Ber Antibiotic carbomycin A shows high activity against a gey’s “Manual of Determinative Bacteriology,' sixth edi 35 number of microorganisms. As previously mentioned, it tion (1948). The characteristics of this strain differ in is particularly noteworthy in its action on mycobacteria some respects from the description of Streptomyces and gram positive microorganisms. It demonstrates some halstedi given by Bergey. Living cultures of the new activity against gram negative microorganisms, but this is strain have been deposited with the Fermentation Divi- of a somewhat lower level. The following table gives the sion of the Northern Regional Research Laboratory at 40 antibiotic spectrum of carbomycin A against a variety of Peoria, Illinois, and have been added to its permanent gram negative and gram positive microorganisms. These collection of microorganisms as NRRL-2331. tests were run by seeding nutrient broth containing various TABLE I Streptomyces halstedi, NRRL-2331

Color Growth Amount of Growth Remarks Aerial Mycelium and Sporulation Soluble Pigment Glucose Asparagine.-----...-- Moderate------Good sporulation. Mouse Gray (R) None.------Edge of colony Smooth, colony slightly to Dark Olive Gray (R); some sec- elevated; reverse white to very light tors of white aerial mycelium. brown; spores gram-positive rods measuring 2.27 X i.95. Some loose Spirals; Single chains curved at end. Dilution plates: Colonies alike except for varying degrees of color from Olive s Gray (R) to Dark Olive Gray (R). Glucose agar------do------White aerial mycelium; no spores; edge -----do------Colony wrinkled. Reverse creamy lichenold. . white. Nutrient agar------Poor to moderate.--White aerial mycelium; some white -----do------Reverse creamy white. sporulation around edge of colony; some waxy colonies. Potato plugs------do------White aerial myceliun, no sporulation- Lig. grayish OW, Calcium malate------do------Good sporulation, Deep Mouse Gray None.----...- Reverse creamy white. (R); spreading colonies. Starch plates------Moderate.------White aerial myceliun, poor sporula------do------Reverse white; poor hydrolysis. tion, grayish white. Synthetic agar------Good------Goodsporulation. Mouse Gray (R) -----do------Reverse dark gray to light gray; spirals to Deep Mouse Gray (R). present, not numerous. Cellulose------Poor------Emerson------Good------On some parts of tube no sporulation, None.------Reverse creamy white. white aerial mycelium; on other parts Mouse Gray (R) sporulation. Dextrose Nitrate Broth- - Moderate. - Nitrates reduced. Gelatin (21 C.).------. - Good-- Pooriquefaction. Reverse white. Skimmed Milk (15 days).---- Moderate. Milk coagulated in 2 tubes; Milk un m changed in 2 tubes. No peptionization. Proteolysis very weak. . . . 2,796,879 - - 3 4 concentrations of the pure antibiotic with the particular TABLE IV organism. The figures in the column marked "growth' Activity of carbonycin A against mycobacteria indicate the highest concentration tested (in micrograms/ milliliter) at which growth of the microorganism still occurred. The second column, "no growth,” shows the Incg.fml. ranae phlei snematis 607 berolinense builyricum minimum concentration used at which no growth of the microorganism occurred. The test was conducted under m w standardized conditions, the tubes being incubated for a -- wo- Ward -- t w t standardized time. -- --- c ------re -- TABLE III It will be seen from the above table that carbomycin A displays an unusual degree of activity against these myco mcg.fml.Growth, Nomcg.fml. Growth, 5 bacteria. Antibiotic carbomycin. A has been found to possess a Strep. faecalis.------K. 19 relatively low level of toxicity when used in test animals. Brucella bronchiseptic 6.25 2.5 A. caerogenes 2------50 10) For instance, the LD0 value, when the antibiotic is ad E. coli 2------100 ------ministered intravenously to mice, as a solution in acidified Proteus Sp. ------50 100 Pe. aeruginosa. 173------50 00 20 water is approximately 7 mg/20-gram per mouse. Toxic M. albicans 8------i00 ------ity to other species and by other routes of administration E. typhosa. 344.---- 50 00 R. pneumonide 132- 50 O) is comparable. S. paratyphi A. 134- 50 100 This invention includes, within its scope, a process for S. paraiyphi.B. 139-- 50 100 Micrococcits pyogenes war. aureats 5- 0.9 0.39 growing the hitherto undescribed strain of S. halstedi. B. subtilis 219.------0.39 0.73 25 The cultivation of the microorganism preferably takes place in aqueous nutrient media at a temperature of about 24-30 C, and under submerged conditions of agita tion and aeration. Nutrient media which are useful for In addition to the above microorganisms, certain resist this process include a carbohydrate, such as sugars, starch, ant strains were used in order to determine the activity of 30 glycerol, corn meal, and a source of organic nitrogen, such the new antibiotic. For instance, using an aureomycin as casein, soybean meal, peanut meal, wheat gluten, cot and chloramphenicol resistant strain of A. aerogenes, it tonseed meal, lactalbumin, enzymatic digest of casein, was found that growth occurred at a concentration of 25 tryptone. A source of growth substances, such as dis micrograms of carbomycin A per milliliter of nutrient tillers' solubles, yeast extract, molasses fermentation resi broth, but that no growth occurred at a concentration of 35 dues, as well as mineral salts, such as sodium chloride, 50 micrograms/milliliter. In a second test of this nature, potassium phosphate, sodium nitrate, magnesium sulfate a strain of A. aerogenes resistant to both streptomycin and and trace minerals such as copper, Zinc and iron may also streptothricin was also found to grow in the presence of be utilized with desirable results. If excessive foaming is 25 mcg. of carbomycin. A per ml. but it did not grow at encountered during the fermentation, anti-foaming agents, a concentration of 50 mcg../ml. In addition to showing 40 Such as vegetable oils, may be added to the fermentation medium. The pH of the fermentation tends to remain the effectiveness of the new antibiotic against such resist rather constant, but, if variations are encountered, a buf ant strains, this test also demonstrated that the antibiotic fering agent, such as calcium carbonate may also be added definitely differs from aureomycin, chloramphenicol, strep to the medium. tothricin, and streptomycin. It will also be noted that 45 Inoculum for the preparation of antibiotic carbomycin antibiotic carbomycin A is even more active against anti A by the growth of the new strain of S. halstedi may be biotic-resistant strains of A. aerogenes than it is against obtained by employing growth from slants on such media the normal strain of A. aerogenes, as indicated in the as Emerson's agar or beef lactose. The growth may be above Table III. A strain of Staphylococcus aureus was used to inoculate either shaken flasks or inoculum tanks found to be resistant in vitro to the action of penicillin 50 for submerged growth, or, alternatively, the inoculum tanks at a concentration of 100 units/ml.; dihydrostreptomycin may be seeded from the shaken flasks. The growth of at 100 units/ml.; polymyxin at 100 units/ml.; and sensi the microorganism usually reaches its maximum in about tive to the action of chloramphenicol at 100 mcg../mi. and two to three days. However, variations in the equipment aureomycin at 12.5 mcg../ml. This organism was in used, the rate of aeration, rate of stirring and so forth may 55 effect the speed with which the maximum activity is hibited by a concentration of 3.1 mcg/ml. of carbo reached. In general, from about 24 hours to four days mycin A. is a desirable period for producing the antibiotic. Aera Antibiotic carbomycin A has been shown to be highly tion of the medium in tanks for submerged growth is effective against a variety of types of mycobacteria. In maintained at the rate of about one-half to two volumes of the following table are given the results of such tests. In 80 free air per volume of broth per minute. Agitation may this case tubes of Dubos liquid medium, containing 0.5% be maintained by suitable types of agitators generally albumin, were used as the test medium. Antibiotic car familiar to those in the fermentation industry. Aseptic bomycin A was added to the medium at various levels as conditions must, of course, be maintained throughout indicated in the first column of the following table. Tubes the transfer of the inoculum and throughout the growth of the medium containing various concentrations of the 5 of the microorganism. antibiotic, as indicated, were then seeded with cultures of After growth of the microorganism, the mycelium, the various mycobacteria. The identity of the species is which is generally quite luxuriant and fine, may be re moved from the fermentation broth by various standard indicated at the head of each of the columns in the table. equipment, such as filter-presses, centrifuges, and so forth. Under the name of the species are indicated the results of 70 It has been found that reducing the pH of the whole fer the test. It was conducted by incubating the tubes under mentation broth to about 3.0 assists in the filtration. sterile conditions for 18 hours. Lack of growth in the Since the antibiotic is not highly stable under acid con tubes at the end of the standard test period is indicated by ditions, the fermentation broth should be adjusted to ap a minus (-) sign, doubtful growth is indicated by h and proximately neutrality once it has been filtered. The growth is indicated by --. s carbomycin A may be recovered from fermentation broth 2,796,879 5 6 by several different procedures. Alternatively, the whole the infra-red region. Among these are the following fre broth may be used as is or it may be dried. The anti quencies (in reciprocal centimeters): 3510, 2920, 1732, biotic may be further purified by various means; for in 1690, 1630, 1460, 1375, 1300, 1230, 1160, 1122, 1077, stance, the compound may be extracted from aqueous so 1052, 1025, 1015, 976, 916, 910, 873, 860 and 837. An lution at neutral or slightly alkaline pHs, preferably be infra-red spectrum of such a chloroform solution is shown tween about 6 and about 10, by means of a variety of in Fig. 1 of the attached drawings. water-immiscible organic solvents, including ethers, aro Salts of carbomycin A with acids may be prepared by matic hydrocarbons, esters, ketones, lower alcohols and treatment of the base with the appropriate acid. For in halogenated hydrocarbons. Examples of these are diethyl stance, if the base is dissolved in ether and a stream of ether, benzene, toluene, ethyl acetate, butyl acetate, methyl 10 hydrogen chloride is bubbled into the solution, carbomycin isobutyl ketone, butanol, and chloroform. Even at acidic A hydrochloride immediately precipitates. Other acids pH's some of these solvents extract an appreciable amount may be used in the same or similar processes to make w of antibiotic. This is particularly true of the water other acid salts of carbomycin A. However, as pointed immiscible alcohols, such as butanol, pentanols, and so out above, carbomycin A is not highly stable in aqueous forth. The antibiotic may be extracted from most solvent 5 acid solutions, so care must be exercised. - solutions back into acidified water, preferably at a pH of A number of different microorganisms may be used to below about 2.5. If desired, the solvent extract may be determine the potency of carbomycin A. preparations. concentrated before extraction into acidified water. By These include K. pneumoniae, B. subtilis, Staph. aureus, adjustment of the pH to neutrality or alkalinity, the anti and other standard test organisms. When K. pneu biotic may be re-extracted into one of the solvents indi moniae is used, it may be in the form of a turbidimetric cated above. Upon drying the solvent and concentrating test or a plate assay. Crystalline chloroamphenicol may the solution the antibiotic crystallizes. It generally crys be used as a standard, assigning a value of 1000 units/mg. tallizes as white, blunt needle crystals. However, in some to this antibiotic. On this basis, crystalline carbomycin cases a plate-like form of the crystals is obtained. The A has a potency of 750 to 850 chloroamphenicol units product may be recrystallized from benzene, ether, lower per mg. In the preparation of carbomycin A by fermenta ketones, or lower alcohols. If desired, a solution in one tion, broths having a potency of about 15 to about 100 of the water-miscible alcohols or ketones is concentrated units/ml. are obtained. and water is added to assist the crystallization. Alter Carbomycin A has been distinguished from other anti natively, the antibiotic may be recovered by adsorption biotics by its properties. The properties that have been from broth or concentrates on activated carbon. Elution 30 used for this comparison include those listed above, as with acid or with water and a water-miscible solvent con well as measurements by means of paper chromatography, taining acid removes the compound in a purified form. which show a distinct difference. Comparisons have also Carbomycin A is a weakly basic, white, organic com been made with microorganisms resistant to various other pound that is soluble in dilute aqueous acids, but only antibiotics. slightly soluble in water (approximately 0.5 mg/ml.). It The following examples are given by way of illustration is extremely soluble in a number of organic solvents, and are not to be considered as the only manner in which such as acetone, ether, benzene and chloroform, and is this invention may be embodied. It is to be understood somewhat less soluble in methanol and ethanol. It is that protection hereof is only to be limited by the specific insoluble in hexane. On concentrating a solvent solution wording of the appended claims. of carbomycin A, there is a tendency to form super-satu 40 EXAMPLE I rated solutions, and the crystals which form tend to re tain some of the solvent used. The compound has de Spores of an Emerson agar slant of S. halstedii creased stability in strongly acid and in basic solutions, NRRL-2331 were transferred under aseptic conditions to even at room temperature. It is quite unstable on heat a nutrient medium having the following composition: ing in acid solutions and on heating in alkaline solutions, 45 Grams and shows its maximum stability in a pH range of from Cerelose ------10 3 to 7. Its stability in the dry state or dissolved in dry Soybean meal------10 solvents is good. Sodium chloride------5 Highly purified, crystalline carbomycin. A base, such Distillers' solubles------5 as is attained by recrystallization from lower alcohols, 50 Calcium carbonate------1. ether, benzene and like solvents, tends to occur in the form of solvated crystals from which the solvent may be re This mixture was diluted to one liter and adjusted to pH moved more or less completely by prolonged drying at 7 with potassium hydroxide before sterilizing. After the 100 C. Such dried material has a melting point of medium had cooled, the spores were added. The or 217-218 C. with slight decomposition (determined by 55 ganism was grown in shaken flasks for two days at about inserting the sample in the melting point bath at 196° C. 28° C. The broth containing the mycelium was then and raising the bath temperature at a rate of 3.6 per transferred to twenty times its volume of a sterile medium minute). The crystalline anhydrous material forms a of the following composition: monohydrate when exposed to atmospheric moisture. Grams The anhydrous crystalline base contains the elements car 60 Starch ------20 bon, hydrogen, nitrogen and oxygen in the following pro Casein ------10 portions by weight: Soybean meal------15 Percent Calcium carbonate------1. Carbon ------59.8 This mixture was diluted to one liter and adjusted to pH Hydrogen ------8.2 65 7 before autoclaving and seeding. After agitation and Nitrogen ------1.7 aeration under sterile conditions for two days, the poten Oxygen (by difference).------30.3 cy of the broth was found to be 100 units/ml. The The material has an optical rotation of Iale-58.6 (in mixture was adjusted to pH 3.0 and the mycelium was dilute chloroform solutions). It was found to have a removed by filtration. The filtrate was adjusted to pH pKa value of 6.4 by conventional titration and a molecul 70 6.5, and it was then extracted twice with one-quarter vol lar weight of approximately 866. The ultra-violet ab ume of methyl isobutyl ketone. The combined solvent sorption spectrum of the pure base possesses a definite phases were concentrated to one-tenth volume under vacu peak at 242 mu (10 mg. in 250 ml. of 0.1 N-hydrochloric um. The antibiotic was then extracted into water at a acid). The anhydrous crystalline base, when dissolved pH of about 2.0, using sulfuric acid to adjust the pH. in chloroform, shows a number of characteristic peaks in 75 The aqueous phase was separated, washed with a small 2,796,879 7 3 volume of benzene to remove dissolved methyl isobutyl the broth and solvent extracting it at a pH between about ketone, and the aqueous phase was adjusted to pH 6.5. 6 and about 10. The antibiotic was extracted several times with ether and 5. A substance effective inhibiting the growth of gram the separate ether phases were dried over anhydrous so positive bacteria and mycobacteria, selected from the dium sulfate. The solvent was removed by distillation 5 group consisting of a basic substance slightly soluble in and the antibiotic crystallized as white needles. The water, very soluble in acetone, ether, benzene and chloro product was recrystallized by dissolving it in acetone, form, and capable of forming salts with acids; whose filtering to remove solid impurities, concentrating, and anhydrous crystalline base (a) contains the elements car adding water. A flow sheet illustrating the above ex bon, hydrogen, nitrogen and oxygen in the following pro traction process is shown in Fig. 2. The product obtained 10 portions by weight: was found highly effective against a variety of myco Percent bacteria and gram positive microorganisms. Carbon ------59.8 EXAMPLE II Hydrogen ------8.2 A medium was prepared having the following com Nitrogen ------1.7 position: 5 Oxygen (by difference).------30.3 Grams (b) has an optical rotation of Iol--58.6 (in dilute Starch ------20 chloroform solutions), (c) possess an ultra-violet ab Distillers' solubles------10 sorption spectrum showing a definite peak at 242 mu, Soybean meal------15 (in dilute HCl), and (d), when dissolved in chloroform, The mixture of materials was added to one liter of water exhibits characteristic absorption peaks in the infra-red and the pH was adjusted to 7 before the addition of one region at the following frequencies expressed in recipro gram of calcium carbonate. The medium was then auto cal centimeters: 3510, 2920, 1732, 1690, 1630, 1460, claved and seeded with S. halstedi NRRL-2331 inocu 1375, 1300, 1230, 1160, 1122, 1077, 1052, 1025, 1015, lum under sterile conditions. The mixture was subjected 25 976, 916, 910, 873, 860 and 837; and the acid salts of to aeration and agitation under sterile conditions at about said basic substance. 28° C. for two days. The filtered broth assayed 25 units 6. The basic substance defined in claim 5. of carbomycin A per milliliter of solution. 7. An acid salt of the basic substance defined in What is claimed is: claim 5. 1. A process for producing carbomycin A, which com 30 8. A hydrochloride of the basic substance defined in prises cultivating Streptomyces haistedi NRRL-2331 in claim 5. an aqueous nutrient medium under submerged aerobic conditions until substantial antibacterial activity is im References Cited in the file of this patent parted to said medium. UNITED STATES PATENTS 2. A process as claimed in claim 1 wherein the carbo 35 2,516,080 Sobin et al. ------July 18, 1950 mycin A is recovered from the fermentation broth. 3. A process for producing carbomycin A, which com OTHER REFERENCES prises cultivating Streptomyces halstedi NRRL-2331 in Gardner et al.: Brit. J. Exptl. Path., 1942, vol. 23, pp. an aqueous nutrient medium under agitated, submerged 123 to 127. aerobic conditions at a temperature of from about 24 40 Kocholaty et al.: J. Biol. Chem., May 1947, pp. 765 to about 30° C. for a period of from about one day to 769. about four days. Waksman: “The Actinomycetes,” pub. 1950, pp. 116 4. A process as claimed in claim 1 wherein carbomycin 119. ‘. . A is recovered from the fermentation broth by filtering