IL38693A - Antibiotic a-4696 - Google Patents

Description

NOVEL ANTIBIOTIC A-4696 4696-A στπ *DT:J*¾3R ηοιπ T e present: invention provides novel antibiotic A- 696 and the pharmaceutically acceptable acid addition salts thereof.

Tooth decay and gum disease are among the important health problems with which man is continuously struggling. The evidence is convincing that dentobacterlal plaques are conducive to tooth caries, or periodontal lesions, or both. There is a need for prophylactic program which will control the formation of dentobacterlal plaque or keep such deposits below the level at which toxic reactions occur. Antibiotics which inhibit the growth of plaque-forming microorganisms have been developed, but there remai s a need for more effective agents useful in the prevention and treatment of tooth decay and gum disease, The efficient production of animal proteins for human consumption is a continuing problem in agriculture. Many agents h¾ve been found, among them numerous antibiotics, which are effective as feed additives in producing additionffl. weight gain in growing chickens and swine.

There is a never ending need for improved materials which are safe and economic and can be added to animal feed to increase the weight gain an to Improve the feed effl^. c, ciency. The discovery of agents which will accomplish this purpose epresents a real advance in the art.

The antibiotic of this invention is produced by culturing the organism Actinoplanes sp,, strain ATCC 233 2, in an aqueous nutrient medium under submerged aerobic fermentation conditions. The antibiotic is sepa-rated from the filtered fermentation broth by adsorption onto an activated adsorbent and eluted therefrom with an acidic solvent. Antibiotic A-4696 is purified s a crystalline compound by adsorption on an acidified chromatographic adsorbent and eluted therefrom with an acidic solvent. Preferably, the antibiotic Is converted to the hydrochloride or sulfate salt in an aqueous methanol solu-t ion and obtained as a pure crystalline salt by adding acetone thereto and filtering off the precipitate that forms, Antibiotic A-4696 possesses antibacterial and growth promptant activity.

The novel antibiotic of this invention is a basic compound capable of forming salts with suitable acids. The characterization data presented below are for antibiotic A-4696 in the form of its hydrochloride salt. The antibiotic is conveniently isolated and characterised as the hydrochloride salt, although other pharmaceutically acceptable salts can be prepare^ by employing methods well-known in the art.

Antibiotic A-4696, as the hydrochloride salt, is a white crystalline compound with a melting point greater than 220°C, It is soluble in water, and insoluble in solvents such as' methanol, acetone, ether, chloroform, pyridine, benzene, aliphatic hydrocarbons, and the like. It is very stable in solution over a pH range of from about 1.0 to about 10.0, at temperatures up to about 27°C.

E!ectrometric titration of A-4696 hydrochloride in water or in dimethylformamide :water (2:1) produces a curve approximating a straight line with a slope of about 0.14 from pH 6.0 to 13.0.

An average of several microanalyses has shown A-4696 hydrochloride to have approximately the following percent elemental compositions: C, 51.33; H, 5«79i , .... N, 5,46; 0, 30.96; CI, 6.7? percent. The apparent molecu lar weight as determined by the vapor pressure osmotic method is II58, The specific rotation ( a_7D), of A-4696 hydrochloride at 25°C. is -42.3° (0=1, Hg0) .

The ultraviolet absorption spectrum of A-4696 hydrochloride in acidic and neutral solutions shows a single absorption maximum at 276 nw. , with an extinction coefficient, of 45. In a basic solution A-4696 1cm. hydrochloride exhibits a single absorption maximum of 300 mu. , with an extinction coefficient, E^m of 6 .

The infrared absorption spectrum of A-4696 hydrochloride in a mineral oil mull is shown in Figure 1 of the accompanying drawing. The observed distinguishable absorption maxima over the range 2.0 to 15.0 Λ. are as follows: 3.0, 5.8, 5.9, 6.03, 6.15, 6.28, 6.63, 6.85, 7.27, 7.75, 8.1, 8.25, 8.9, 9.4, 9.9, 10.1 microns.

The paper c romatographic profile of A-4696 hydrochloride is shown by the values in Table I, below. The values were obtained in each instance using Whatman No. 1 paper and the indicated solvent system. The location of the antibiotic on the chromatogram was determined by bloautograph using Bacillus subtilis as the organism.

TABLE I Paper Chromatography of k-UbVb Hydrochloride Solvent System R Values 1 0.88 2 0.72 3 0.80 0.59 0.35 6 0.77 7 0.80 8 0.7^ 9 0.87 0.59 11 0.77 *Rf value is defined as the ratio of the distance traveled by the antibiotic from the origin to the distance traveled by the solvent front from the origin.

Key to Solvent Systems: 1. Water saturated with butanol plus 1% p-toluenesulfonic acid. 2. Water saturated with methyllsobutyl ketone plus 1$ Ρτ toluenesulf onlc acid. 3. Water saturated with methyllsobutyl ketone plus 1$ p- toluenesulf onlc acid and 1# plperidlne.

. Water: met anol: acetone (12;3:l). The solution is adjusted to pH 10.5 with NH^OH and then lowered to pH . Methanol: 0. IN HCl (3:1). 6. One percent methylisobutyJ. ketone plus Q .5^ NH^QH In water. 7. # ft ·' ¾ Seven percent sodium chloride plus 2. methyllsobutyl ketone In water. 8. Ten percent propanol In water, 9. Butanol.ethanol: ater (150: 15 : 13.5) .

. Propanol: pyridine: acetic acid:water (15 : 10: 3 : 12 ) . 11. Water Methanol: acetic acid (70: 24 : 6 ) .

The pharmaceutically acceptable salts of antibiotic A -4696 can be prepared with mineral acids such as hydrochloric, hydrobromic, sulfuric, phosphoric, and the like, and also with organic acids such as citric, tartaric, maleic, p-toluenesulfonic, salicylic, fumaric, acetic, propionic, and the like. The antibiotic salts of such acids can be prepared, for example, by acidifying a solution of the antibiotic free-base with the desired acid and precipitating the salt thusly formed by introducing ten volumes of acetone to the solution containing the A -4696 acid salt. The salts can likewise be prepared in certain instances by ion exchange on an ion exchange column.

Other commonly used methods for the preparation of antibiotic salts can also be employed.

The novel antibiotic of this invention has an inhibiting action against the growth of many microbial organisms which are pathogenic to man, animals and plant-life, and is, therefore, useful in suppressing the growth of such organisms. The levels at which A -4696 hydrochloride shows inhibition against the growth of the illustrative organisms are set forth numerically in Table II, below. The Inhibition levels were determined by either the agar-dllutlon test or the broth-dllutlon test, and are stated in terms of the minimum inhibitor concentration (MIC), microgram(s) per milliliter (me , /ml. ) . (identic fied in Table II by the letter "ad" and "bd", respectively).

In the agar^-dllution test the test organism is streaked or implanted on agar plates containing various concentrations of A-4696 hydrochloride in the agar. The test plates are incubated at 37°C. for 48 hours, and the MIC is determined as the plate at the lowest concentration of the antibiotic where growth pf the test organism is inhibited.

In the broth-dilution test a series of tubes cpntaining nutrient brpth and varying cpncentratipns of A-4696 hydrochloride are inoculated with the test organism and Incubated at 37°C. for 24 hours. The MIC is determined as the lowest antibiotic concentration where no growth is present in the tube.

TABLE II Minimum Inhibitory 1 concentration Test Organism (meg./ml. ) Staphylococcus aureus 3055 12 .5 ad 6.25 bd Bacillus subtills 0.78 ad Mycobacterium avium 0 , ad Streptococcus faecalis 3 .12 ad Trichophyton mentag o hytes P .2 ad Vibrio coli 12 .5 bd Mycoplasma gallisepticum 50.0 bd Staphylococcus aureus (penicillin resistant ) 10. 0 bd Staphylococcus aureus (methicillin resistant ) 5.0 bd Diplococcus pneumoniae 3 . 12 bd Clostridium perfringens 1.25 bd Clostridium tetani 2 .5 bd Corynebacterium gravis 1.25 bd Lactobacillus casei ioo ad Leuconostoc citrovorum >100 ad Escherichia coli 7100 ad Proteus sp. >ioo ad Pseudomonas sp. >100 ad Salmonella sp. >100 ad Vibrio metschnllovii 100 ad Saccharomyces pastorianus >100 ad Candida albicans >100 ad Antibiotic A^46 6 hydrochloride when given by subcutaneous injection to mice has in vivo antimicrobial activity against infectious organisms; for example: the values (effective dose to protect 50 of the test animals) in illustrative infections are as follows when two doses are employed: Staphylococcus aureus, 9.2 mg./kg.; Streptococcus pyogenes, 0.68 mg./kg.; and Dlplococcus pneumoniae, 0.7,6 mg./kg.

The novel antibiotic of this Invention is also, effective in inhibiting the growth of microorganisms which contribute to the development of periodontal disease and tooth decay. For example, a solution of A- 696 hydros chloride exhibits antimicrobial activity against plaque forming organisms as ^illustrated by the following test system: Tubes of nutrient broth containing 5$ sucrose are inoculated with a cariogenic microorganis . Glass rods are immersed in the medium and the tubes are Incubated at 37°C. overnight after which the layer of plaque (primarily cells and dextran) forms on the surface of the rods. The rods are then transferred to solutions containing varying concentrations of A- 696 hydrochloride and allowed to remain in contact with the antibiotic for 5, 10, and 15 minutes. After the appropriate time has elapsed, the rods are rinsed with sterile, deionized water, and immersed in uninoculated medium containing sucrose. After incubating overnight at 37°C. bromthymol blue is added to each medium. Growth is detected by observing the color change from blue to yellow due to the acid production of the organisms when grown in a sucrose containing medium.

A solution of A-46 6 hydrochloride at a concentration of 0.01$ was effective against an unidentified cariogenic Streptococcus sp. when in contact with the plaque encrusted rods for ten minutes. The growth of this organism was prevented by A-4696 hydrochloride at a 0.1$ concentration when the solution was In contact with the test rod for 5 minutes.

Moreover, A- 6 6 hydrochloride Inhibits the growth of the plaque-forming organism Odontomyces ylscosus at a concentration of Ο.25 meg. /ml. In a broth-dilution test.

The incorporation of A-4696 or one of its acid addition salts into an appropriate toothpaste, gel, powder, or the like, or a suitable mouthwash, or other oral hygiene preparation, can provide an effective method for inhibiting the development of dental caries and periodontal disease. Alternatively, a solution of A-4696, or one of of its acid addition salts at an appropriate concentration can be applied to the surface of the gums and teeth with a suitable swab.

Antibiotic A-4696 has also; been found to be effective in promoting growth in animals. When A-4696 hydrochloride is added to the basal ration of growing chicks at a rate of 45.4 gm./ton, the weight gains and the feed efficiencies are shown to be improved over chicks receiving no antibiotic. Table III, below, shows the data from four separate experiments conducted to determine the effectiveness of A-4696 hydrochloride in chicken weight gain tests. All tests were carried out in chick batteries located in quarters maintained at about 25°C. The birds used in the various tests varied, from 1 to 5 days of age at the start of the experiment. Test periods were varied from 10 to 17 days in length. Continuous lights and ad_ libitum feeding were used throughout the trials.

TABLE III Weight Gains and Peed Efficiency in Young Fed A-4696 hydrochloride in the Feed Ra A-4696 Hydrochloride No . of Days .No . of Birds Feed Ration g. /ton of Feed on Treatment in Treatment Basal ration 0 10 40 Basal ration plus 45.4 10 40 Jl-4696 Hydrochloride Basal ration 0 10 20 Basal ration plus 45.4 10 20 A-4696 T¾rdro-chloride Basal ration 0 17 80 Basal ration plus 45. 17 80 A-4696 Hydrochloride Basal ration 0 17 80 Basal ration plus 45.4 17 80 A-4696 Hydrochloride TABLE III (Continued) A- 696 Hydrochloride No. of Days No. of Birds Peed Ration g./ton of Feed on Treatment in Treatment Basal ration 0 17 80 Basal ration plus 45.4 17 80 A -4696 Hydrochloride The effect of feeding A-4696 hydrochloride to weanling pigs and its influence on the average dal ly , weight gain and feed efficiency was tested . Weanling pigs at 4 to 5 weeks of age were maintjalned on a diet which con talned no antibiotics for approximately one week before initiating the experiment . The pigs were then weighed and ass igned to experimental groups on the basis of weight and litter . Pigs were housed in enclosed buildings with adequate heat and ventilatipn, a d feed and water were available ad libitum at all times . In eaclr) test the pigs were divided into two groups, o e of the groups received the basal ration with no antibiotic added, and the . other group received the game basal ration Plus antibiotic .

A-4696 hydrochloride . Table IV, shown below, lists the Improvement in average daily gain and feed efficiency in those pigs which were fed the A-4696 hydroc hloride as contrasted with those which received only the basal ration.

TABLE IV Average Daily Weight Gain and Feed Effic Fed Antibiotic A-4696 Hydrochloride as a A-4696 Hydrochloride No . of Pigs No . of Days Feed Rat ion g . /ton in Test n Test Basal ration 0 11 28 Basal ration plus 20 11 28 A-4696 Hydrochloride Basal ration 0 11 34 Basal ration plus 50 11 3* A-4696 Hydrochloride The acute toxicity of A-4696 hydrochloride, determinated in mice and expressed as I^Q' 1S 2*^°0 nig. /kg. when administered intraperlt oneally .

The novel antibiotic of this invention is produced by culturlng a newly discovered strain of an Actino-planes organism under aerobic conditions in a suitable culture medium until the culture medium contains substantial antibiotic activity. The antibiotic can be recovered by employing various isolation and purification proce-r dures commonly used and understood in the art. When the antibiotic is to be incorporated in feed-istuff for animal use, a lesser degree of purification is required than that which is necessary if the antibiotic is to be used for medicinal purposes.

The microorganism used according to this invention for the production of antibiotic A-4696 has been iden-r tified as a strain of a species of Actjnoplanes of the family Actinoplanaceae . The actinoplanaceae are a new family of microorganisms of the order Ac inomycetales, having been first described by Dr. John N. Couch, Jour.

Ellsha Mitchell Sci., Soc., β£, 315-318 (19^9); and 66, 87-92 (I95O); Trans. New York Acad. Sci., 16, 315-318 (1954); Jour.. Ellsha Mitchell Sci. Soc., 71, 148-155 and 269 (1955); Bergey's Manual of Determinative Bacteriology, 7th Edition, 825-829 (1957); and Jour. Ellsha Mitchell Sci. Soc, 79, 53-70, (1963).

The Actinoplanes sp. useful for the production of antibiotic A-4696 has been deposited and made a part of the stock culture collection of the American Type Culture Collection, Rockvllle, Maryland, from which it is available to the public without restriction under the number ATCC 23342.

The Actinoplanes sp. useful for the production of A-4696 was isolated from a sample of soil obtained from the Cascade mountain area in the state of Washington.

This organism has been designated number 58I in the collection of Dr. John N. Couch at the University of North Carolina.

Actinoplanes sp., strain ATCC 233 2, is charac-terlzed by the physical and cultural properties set forth in the following paragraphs t The system of Rldgeway, Color Standards and Nomenclature, (1912), is employed for the naming of the colors.

Microscopic Morphology and Qeneral Cultural Characteristics of Actinoplanes sp. ATCC 23342 Microscopic Morphology -: Mycelia are rather sparse on Llquidamber (Sweet gum tree) pollen In water. The hyphae are branched, septate, 0.2-1, in diameter. Hyphae fill the pollen grain, extending out in the water to a distance about equal to the diameter of the grain. Sporangiophores project above the surface of the water; sporangial stalks, usually single, at times branched to bear two, or rarely more, sporangia on the same sporangiqphore; stalks 1.0-l.¾^. , thick, septate. Sporangia are rather small, k-i x. in diameter, subglobose, rarely globose, usually with an irregular wall. Mature spores are arranged in one or more Indistinct coils in the sporangium. Sporangial dehiscence is produced by the swelling of an lntersporal substance which causes the sporangium to enlarge and assume an al-most smooth spherical shape. Spores are motile, about 1.0-1.^., in diameter; globose to subglobpse.

Cultural Characteristics on: Czapek Agar (S. A. Waksman, The Actinomycetes, 1950).

Growth is good; the point inoculum about 2 cms. in diameter after eight weeks. The central part is mound-like; the smear flattened and minutely bumpy. The color of the mycelia is a brilliant zinc orange; the agar is pale buff to distinctly yellowish. Sporangia are rarely formed. Peptone Czapek Agar (5 g. of peptone substituted for 2 g. of sodium nitrite). Growth is similar to that observed on Czapek Agar, but furrows, ridges and bumps are more distinct than on Czapek Agar, No sporangia are formed.

As previously noted, Actinoplanes sp., strain ATCC 233^2, can be grown in a culture medium to produce antibiotic A- 69^. T^e culture medium can be any one of a number of different media, However, for economy in production, maximum yield, and ease of isolation of the antibiotic certain culture media are preferred. Thus, for example, starch is one of the preferred sources of carbohy-drate, and soybean meal is one of the preferred nitrogen sources. Other carbohydrate sources which can be used include molasses, glucose, dextrin, glycerol, and the like. Nitrogen sources also Include amino ac d mixtures, peptones, and the like.

Nutrient inorganic salts to be incorporated in the culture media can include the customary salts capable of yielding sodium^ potassium, ammonia, calcium, phosphate, chloride, sulfate, acetate, carbohydrate, and like ions. Additionally, sources of growth factors, such as distillers' solubles and yeast extracts, can be included with beneficial effect on the production of A- 696 antibiotic . As is necessary for the growth and development of other microorganisms, essential trace elements should als o be Included in the culture medium for growing the Actino-planes sp. employed in this invention. Such trace elements are commonly supplied, as impurities incidental to the addition of the other constituents of the medium.

The organism used to produce A- 696 can be grown over a relatively wide pH range . However, it is desirable to adjust the pH. of the culture medium to between about the pH 6.5 and pH 7.2 before lnoc ulation with the organism. As with other Actinomycetes, the pfi of the growing medium gradually changes during the growth period ; the pH at the end of the fermentation period usually ranging from about 7.0 to 7.8.

Submerged aerobic culture conditions are preferred for the production of A- 696. Relatively small amounts of the antibiotic can be produced by shake flask culture; however, for the preparation of large amounts , s ubmerged aerobic culture in sterile tanks is preferred . The culture medium in the sterile tank can be inoculated with a sporulated or mycelial fragment suspension; but because of the time lag experienced when a sporulated suspension is used , the preferred inoculum is the vegetative form of the cult ure . More efficient use of fermentation equipment is realized by avoiding the time lag in the growth cycle . \ Accordingly, it is desirable to produce a vegetative inoculum of the organism by inoculating a relative-ly small quantity of culture medium with the spores or mycelial fragments of the organism, and when a young active vegetative inoculum is obtained aseptically transfer it to the large tank. The medium in which the vegetative inoculum is grown can be the. same a that utilized for large-scale fermentation of A-4696, although other media can be employed.

The A-4696 producing Actlnoplanes sp., strain ATCC 23342, grows at temperatures between about 209 and 40°C„ The largest amounts of A-4696 appear to be produced at a temperature of about 30°Q.

Sterile air is blown through the culture medium in the submerged aerobic culture process. The volume of air sparged into the culture medium varies from about 0,1 to about 1.0 volume of air per minute per volume of culture medium. The most efficient growth and antibiotic production are achieved when the volume of air is at least 1/2 volume of air per minute per volume of culture medium.

The rate of production of A-4696 and the concentration of antibiotic activity in the culture medium can be followed during the growth period by testing samples of the fermentation broth for antibiotic activity against organisms known to be susceptible to the antibiotic. One such assay organism useful in the present invention is Bacillus subtllis . The bioassay can be carried out by the standard turbldimetric or cup-plate methods, or by the paper disc assay on agar plates.

Generally, maximum production of the antibiotic occurs within about 4 to 6 days in shake, flasks or submerged aerobic culture fermentations.

Antibiotic A-4696 can be recovered from the culture medium and separated from other substances which may be present by adsorptlve and extractive techniques. Adsorptlve techniques are preferred because such procedures avoid the use of large volumes of solvents required In extraction processes.

After fermentation, the antibiotic activity is present in both the broth and mycelium. The culture medium is filtered to separate the fermentation broth from the solid mycelia. The mycelial cake is washed with water and then slurried in water adjusted to a pH of 10.5 with 5. ON NaOH, agitated vigorously for 30 minutes and filtered. The two filtrates and the wash water are combined and the antibiotic activity adsorbed therefrom on a suitable adsorptlve agent , such as activated carbon, activated acid alumina, polyamide resin, cellulose ailica gel, and the like. The adsorption can be accomplished by passing the antibiotic activity-containing solution over a bed of the adsorbent, or by adding the adsorbent to the solution, mixing thoroughly for 20 to 30 minutes and filtering off the exhausted solvent. The antibiotic adhering to the adsorbent can be recovered as Impure A-4696 by customary elutlon procedures.

The crude A-4696 can be purified by the preparation of the picrate salt and the conversion thereof to the hydrochloride salt, or by adsorption and elutlon procedures using an appropriate adsorbent or Ion exchange resin. Suitable adsorbents include Polyamide resin (M.

Woelm, Eschwege, Germany), acidic alumina, neutral alumina, basic alumina, silica gel, Amberlite XA -2 (Rohm and Haas, Philadelphia), Amberlite XA - (Rohm and, Haas, : Philadelphia), and Dowex §0 (H+) (Dow Chemical, Midland, Michigan).

This invention is further illustrated by the following examples: EXAMPLES 1 A. Shake Flask Fermentation -of -Antibiotic A- 66 >; Mycelial fragments of Actlnoplanes sp., strain ATCC 23342, were inoculated on a nutrient agar slant having the following composition: Ingred lent Amount Pre-cooked oatmeal 6p g.

Yeast 2.5 g.

KgHPC 1.0 g.

Dried distiller's solubles 5.0 g.

Czapek's mineral stock* 5.0 ml.

Agar 25 g.

Water, deionized 1 1.

♦Czapek's mineral stock has the following composition? KC1 100 g. gSO^'TH O 100 g. eSO H O 2 g.

(Dissolve in two mis. cone. HC1) Water, deionized 1 1.

The slant was inoculated with ATCC 23342 and incubated for 6 days at 30°C. The culture does not normally sporulate on this medium, and it is necessary to macerate the mycelial mat with a flattened, sharpened, inoculating needle in order to increase the number of potential growth centers. The macerated mature qulture was covered with sterile distilled water and scraped caref ully with a sterile rod to obtain a mycelial suspension.

The suspension thus obtained was used to inoculate 100 ml . of a sterile vegetative medium having the f ollowing composition: Ingredient Amount Glucose 5.0 g Dextrin 20.0 g Soybean meal 15. 0 g Yeast extract 2 .5 g, Calcium carbonate 1.0 g.

Wat er, tap 1 1 , The inoculated vegetative medium was grown for 48 hours at 30°C . on a rotary shaker operating at 250 rpm.

Ten ml. of the incubated vegetative medium was inoculated jLnto 100 ml . of 9 sterile "bump" med ium of the same composition as given next above . The thus inoculated "bump" medium was incubated for 24 hours at 30°C . with constant shaking on a rotary shaker operating at 250 rpm.

Four-tenths ml . of the incubated "bump" medium was inoculated into 100 ml . portions of a production medium of the composition shown below contained in 500 ml .

Erlenmeyer flasks, and sterilized at 120°C . for 30 minutes ; Ingredient Percent Dextrose 1.0 Dextrin 3.0 Pe tone 1.5 Soybean meal 0.5 MgS0i|'7¾0 0.2 Molasses, beet awgaxv • 1.

Corn steep liquor 0.5 Betaine I¾HPC-| 0.05 Water, deionized, q.s. 25 ml.

The pH of the medium was adjusted to 7.5 with 5N sodium hydroxide solution before sterilization. After sterilization the pH was approximately 6.9.

The production fermentation was shaken for about 96 hours at a temperature ,of 3Q°C. on a rotary shaker operating at 250 rpm. The pH at the end of the fermentation cycle was about 7.2.

B. 40-liter tank fermentation of antibiotic A-4696 The preparation of the Inoculum proceeded through the incubation of the "bump" medium detailed under section A, abovte. Twenty-five liters of a production medium as outlined above, with .02$ Dow Corning antifoam added, was sterilized by autoc laving at 120°C. for 30 minutes and charged Into a 40 1. fermentation tank. One-hundred milliliters of incubated "bump" medium was Inoculated into the sterile production medium. The inoculated production medium contained in the 40 1. tank was allowed to ferment for 4 days at 30°C. The fermentation was aerated with sterile air in an amount of about one-half volume of air per volume of culture medium per minute. The fermenting production medium was agitated with a mixer utilizing an impeller of a proper size and turning at an appropriate rpm to insure adequate mixing of air with the medium. The pH of the culture medium gradually increased from an initial level of about 6. 9 to about 7.2 as the fermentation proceeded.

C. Isolation of Antibiotic A-4696 The whole broth obtained from an A-4i>96 ferment-atlon, as described above, was filtered with the aid of a commercial filter aid. The filtrate was set aside. The mycelial cake was washed with 32 1. of water and the wash water set aside. The mycelial cake was then suspended in an additional 32 1 . of water and the pH of the mixture adjusted to pH 10, 5 with 5N sodium hydroxide solution. The mycelial cake water suspension was stirred for 45 minutes and the mixture was filtered. This filtrate and the water wash were combined with the original filtrate from the fermentatipn broth and the pH of combined filtrates was adjusted to pH 4 .0 with I^SO^. The acidified combined filtrates was passed through a carbon column utilizing 1 kg. of activated carbon, (Pittsburgh, 12 x 40) , The ac-r tivated carbon column was washed until the effluent was colorless. The A-4696 activity was adsorbed on the carbon column. The A-4696 activity was eluted from the carbon column utilizing a 1$ HgSO^ solution in acetone; H20 (1 : 1 ) . Two liters of the acidified acetone-water solution was sufficient to elute the A r-4696 activity from the carbon column. The eluate containing the A-4696 activity was treated with a saturated barium hydroxide solution, in order to form a precipitate of barl urn sulfate, thus removing the sulfate ions from the solution.

The mixture was filtered and the barium sulfate precipitate was discarded. The filtrate containing the A-4696 activity was concentrated under vacuum to dryness. The resulting residue comprising the A-4696 activity amounted to approximately 80 g.

D. Conversion of A-4696 Activity to Crude A-4696 ¾rdrp~ chloride The approximately 80 g. of A-4696 activity was taken up in a volume of 5 1. of water, and then treated with 500 g„ of activated carbon (Darco G-60, Atlas Chemical, Wilmington, Del.). This mixture was stirred for 1 hour and then filtered. The filtrate was discarded. The carbon filter cake containing the A-4696 activity was washed with 1 1. of water and the water wash was discarded. The carbon filter cake was further washed with 1 ,1. of 0.05N aqueous hydrochloric acid. The acid wash-was' discarded. The washed carbon cake was eluted by stirring 30 minutes with 500 ml. of an aqueous hydrochloride acid-acetone solution (0.05N HC1: acetone Ti.37) . The mixture was filtered and the filtrate set aside. The elutipn of the activated carbon was repeated 4 times in the same, manner, each time setting aside the filtrate. The five elu-ates containing the A-4696 activity were combined. The combined eluates were then concentrated under vacuum to a volume of approximately 100 ml. Two-hundred milliliters of methanol was added to the aqueous concentrate containing the A-4696 activity. Two liters of acetone was added to this aqueous -methanol solution. A precipitate, consist- -2 - ing of crude A-4696 hydrochloride, formed in the acetone-aqueous methanol solution. After filtering and drying the precipitate, 60.9 g., of crude A-4696 hydrochloride was recovered.

E. Preparation of Crystalline A-4696 hydrochloride.

Twenty-five grams of A-4696 hydrochloride, prer pared according to the procedure outlined in Section D of this example, was dissolved in 20 ml. of water. The A-4696 hydrochloride solution was passed over a water washed Polyamide resin bed, (M. Woelm, Eschwege, Germany), contained in a glass column of approximately 7 x 60 centimeters. The effluent was set aside. The Polyamide resin column was washed with water at a flow rate of approximately 8-10 ml. per minute. The antimicrobial activity of the column effluent was measured by conventional procedures. The effluents containing antimicrobial activity were combined and concentrated to dryness under vacuum. The residue was dissolved in a mixture of 25 ml. water and 50 ml. methanol. This aqueous methanol solution of A-4696 hydro-chloride was acidified to a pH of 2 .0 with 5N HC1. Approximately 1.5 1. of acetone was added to the aqueous methanol solution to precipitate the hydrochloride salt of A-4696. The mixture was filtered and the A-4696 hydrochloride precipitate recovered therefrom.

The filter cake containing the A-4696 hydrochloride was dissolved in minimum quantity of water. An amount of ethanol equal to twice the, volume of water was then added and the mixture was heated to approximately 60°C„ The mixture was then cooled and the hydrochloride salt of A-4696 crystallized therefrom. The crystals were filtered and dried. Approximately 9 g.. of crystalline A-4696 hydrochloride was recovered.

EXAMPLE 2 Purification of A -4696 Over Acidi-Washed Alumina Two and three-tenths grams of crude A-4696 , obtained as described in Example 1 through section D above, was dissolved in 20 ml. of 50 percent aqueous methanol and run over a 2 x 4 cm. column of acid-washed alumina (Alcoa). The column was immediately washed with methanol. The ac-tivity was eluted from the acid alumina with 50$ aqueous methanol. Aliquots of the elution were collected and the antimicrobial activity determined on each fraction. The fractions which exhibited antimicrobial activity were combined, concentrated to approximately 100 ml,. and acetone added to precipitate the A-4696 , The precipitate was filtered and dried in vacuo, yielding approximately 930 mg. of pure crystalline A-4696.

The acid-washed alumina used in the purification described above was prepared as follows: Alcoa activated alumina P^20 was washed with water adjusted to pH 3 with HgSO^ by stirring for 6 hours maintaining the pH at 3.0. The alumina was filtered off, washed with $ aqueous methanol, and dried in an oven at 100°C.

EXAMPLE 3 Purification of A-4696 by the Preparation of the Picrate Salt and Conversion thereof to the Hydrochloride Salt Five-hundred milligrams of crude A-4696, obtained as described in Example 1 through section D above, was dissolved in 2 ml. of water and 25 ml. of a saturated aqueous solution of picric acid were added with stirring. The mix-, ture was allowed to stand overnight at 5°c. and a precipitate formed. The precipitate was centrifuged off and dried. The precipitate thus obtained was dissolved in 2 ml. methanol, the pH was adjusted to 1 .5 with HC1, and added to 500 ml. of diethyl ether to precipitate the A-4696 hydrochloride salt. The hydrochloride salt was centrifuged off, washed with diethyl ether and dried in vacup yielding approximately 305 mg. of A-4696 hydrochloride as a white crystalline salt.

EXAMPLE 4, Preparation of A-4696 Sulfate Salt Two grams of A-4696 hydrochloride, obtained as described in Example 1 through section E above, was dissolved in 300 ml. of water, the pH adjusted to 7.5 with 5N sodium hydroxide, and 30 g. of Darco G-60 carbon was added. The mixture was stirred for 30 minutes; filtered with the aid of a commercial filter aid, and the filter cake washed successively with 300 ml. each of water and 0.O5N HgSO^.

The activity was eluted from the filter cake by adding the filter cake to 500 ml. of a mixture consisting of 1 O.05N HgSOij and 30 acetone, stirring for 30 minutes and filtering to remove the carbon. The filtrate was concentrated to approximately 10 milliliters. About 20 ml. of methanol was added to the concentrate and the thus formed solution was added to 600 ml. of acetone to precipitate the A-4696 sulfate. The precipitate was filtered off; washed with acetone and dried in vacuo, yielding 862 mg. of A-4696 , sulfate as a white crystalline salt.

Claims (8)

1. The antibiotic substance A-4696, or a pharmaceutically acceptable acid addition salt thereof -2-.-— The--anti-b-ieti-o--ef--o-laiflj--l-which in the form of its hydrochloride salt is a white, crystalline substance soluble in water and insoluble in the common or ganic solvents ; has a melting point greater than ,220°C ; has an eiectrometrlc titration curve in 66 percent aqueous dimethylformamlde approximating a straight line with a s lope of about 0. 14 from pH 6.0 to pH 13 .0; has the approximate composition of 51.33 percent carbon, 5.79 percent hydrogen, 5. 6 percent nitrogen, 30.96 percent oxygen, and 6.72 percent chlorine; has an apparent molecular weight of 1158, as determined by the vapor pressure osmotic method ; has a specific rotation, T^J^^^ -<-42..30 (÷(c-=l, HgO) ; has an ultraviolet absorption spectrum, in acidic and neutral solutions at 276 rn^. , with an extinction coefficient, E^m of , and in a basic solution a single absorption maximum of 300 m t. with an extinct ion coeffic ient E^ra of 65 ; and has the following distinguishable bands in its infrared absorption spectrum when determined in a mineral oil mull: 3.0, 5.8, 5.9, 6.03, 6.15, 6.28, 6.63, 6.85, 7.27, 7.75, 8. 1, 8.25, 8.9, 9.4 , 9.9, and 10. 1 microns

2. --3, The hydrochloride salt of antibiotic A-4696 as defined in claim 1.

3. . ---4-, The sulfate salt of antibiotic A-4696 as defined in claim 1.

4. . -5,- A method of producing the antibiotic of claim 1 which comprises cultivating the organism Actinoplanes sp. , strain ATCC 3342, in a liquid culture medium containing assimilable sources of carbon, nitrogen and Inorganic salts under submerged aerobic conditions until a substantial amount , of antibiotic activity is ' imparted by said organism to said culture medium.

5. -6, The method of elaim compr i jW-7 sorbing the antibiotic acitivity produced ont o an adsor-bent ; e luting said antibiotic activity from said adsorbent with an acidic solvent ; adsorbing said antibiotic activity on an acidified chromatographic adsorbent ; and elutlng said antibiotic from said chromatographic adsorbent .

6. .7,, A composition for inhibiting the growth of microorganisms which contribute to -the development of periodontal disease and tooth decay comprising the antibiotic of any one of claims 1-$ and a suitable carrier .

7. -&» An animal feed composition comprising the antibiotic of any one of claims 1- and a s uitable carrier .

8. --9-.- poultry feed composition comprising the antibiotic of any one of claims 1- and a suitable carrier . S. HOROWITZ / CO. AGENTS FOR APPLICANTS