IL32476A - Antibiotic 66-40 and method for its production - Google Patents

Description

ΑΝΤΙΘΙΟΤΙΟ 66-40 AND METHOD FOR ITS PRODUOTION insane >irtB»wi 66-40 »oiK»a»OJK "win This invention relates to a new broad spectrum antibiotic designated Antibiotic 66-40, having an adverse effect upon the growth of gram positive and gram' negative bacteria to a microbiological method of its production, and to pharmaceutical compositions containing such antibiotic. The antibiotic may be prepared, isolated and used in its free form or in the form of its pharmaceutically acceptable functional derivatives.

Antibiotic 66-40 is a biosynthetic elaborate obtained by cultivating an Antibiotic 66-40 producing strain of Micromonospora in an aqueous nutrient medium.

The preferred microorganism used according to this invention for the production of the new antibiotic has been named Micromonospora inyoensis (sometimes hereinafter referred to as M. inyoensis). This species was isolated from a soil sample taken from the Inyo National Forest in the White Mountains of California. One of its strain characteristics is its ability to produce Antibiotic 66-40. A culture of the living organism has been made a part of the permanent collection of The Northern Utilization and Research Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, where it has been assigned accession number NRRL 3292. Sub-cultures of M. inyoensis NRRL 3292 are readily available to the public from the foregoing Agen cy upon request. M. inyoensis is aerobic and grows well on the surface of a variety of solid and liquid nutrient media. It exhibits especially good growth and antibiotic production under submerged aerobic conditions.

M. inyoensis is differentiable from other apecies of Micromonospora by a variety of taxonomical parameters.

After 14 days of incubation at 24-26 C on an agar medium comprising NZ Amine Type A (Sheffield Chemical Company, Norwich, New York), 1% dextrose and 1.5$ agar, growth is observed to be only fair to poor. Macro-scopically, there is no apparent aerial mycelium.

Occasionally a few well developed colonies appear late in the inoculation area. On some plates, a faint reddish brown diffusible pigment associated with the colonies is observed. [In describing the color formations for this observation and others, the following system and references are employed: The color designations consist' of two designators. The first is a color name taken from the "Descriptive Color Name Dictionary" by Taylor, Knoche and Granville, published in 1950 by the Container Corporation of America, USA, with a color chip number corresponding to the color name, the chip number being taken from "The Color Harmony Manual", 4th Edition, published 19 8 by The Container Corporation of America. The second designator consists of a color name and number which refers to the synonym or near synonym found in the National Bureau of Standards (USA), Circular 55j5> November 1, 1955.]The colony surface varies in color from Tile Red g5ne - Strong Brown 55 to Brown Mahogany m6pi. Microscopically the mycelium is long, branched, regular and non-septate as observed by phase contrast microscopy. The mycelium has a diameter of approximately 0.5/U. The spores are borne singly on simple sporophores of 1.0 to 1.5/1 in diameter. The spores are rough walled and ovoid to spherical in shape.

M. inyoensis grows well at 28-37°C no growth occurs at 50°C. On glucose asparagine-agar medium growth is poor. A growing colony of M. inyoensis will hydrolyze gelatin, milk, starch and reduce nitrate to nitrite when such tests are applied as according to Gordon et al.

- J. Bacteriology 69, 1 7 ( 1956 ) and 73, 15 ( 1957 ) .

Additionally, sucrose is utilizab'le as a carbon source Additional culture characteristics of M. inyoensis are set forth in Table A: TABLE A Medium Characteristics Bennett's Agar Growth moderate - Color; rust brown-g5pgj strong brown-55 Emerson's Agar Growth fair - Color; brick red-g6ng moderate reddish brown- 3 Tomato Paste-Oatmeal Agar Growth fair Glucose Asparagine Agar Growth poor Glucose Yeast Extract Agar Growth good - Color: rose brown-g7ni Dark greyish red- 20 (Faint maroon diffusible pigment produced by some colonies. ) Potato slice Growth poor but improved when reagent grade calcium carbonate is added.

Czapek's Agar Growth fair Tyrosine Agar Growth fair, no diffusible pigment Peptone Iron Agar Growth fair, no diffusible pigment M. inyoensis is capable of utilizing a variety of carbon and nitrogen sources. In Table B there is set forth observations on carbohydrate utilization. A visual estimate of the degree of growth is observed in a medium consisting of 0. yeast extract, carbohydrate and 1. 5$ agar all in distilled water.

TABLE B CARBOHYDRATE UTILIZATION Carbohydrate in Medium Growth control poor L-arabinose poor D-glucose good D-galactose fair to poor /3-lactose fair to poor D-levulose poor raffinose poor L-rhamnose poor starch good sucrose good D-xylose fair to poor inositol poor D-mannitol poor d(-)-arabinose poor dulcitol poor D-ribose fair -melibiose poor D(+)-melizitose poor glycerol poor In Table C, nitrogen utilization is set forth as determined by visual estimate of growth on agar plate in a medium consisting of 1% glucose, 1.5$ agar, and nitrogen source as indicated, all in distilled water.

TABLE 0 Nitrogen Source Growth 1# H Amine Type A Fair to moderate 0.5# yeast good 1 asparagine poor 1$ glutamic acid poor 1$ ammonium nitrate poor Antibiotic 66-40, the new compound of this invention, is produced when the elaborating microorganism, M. inyoenals is grown in an aqueous nutrient medium under aerobic preferably submerged conditions as described hereinafter* Antibiotic 66-40 is a basic pseudo-oligosaccharide which is readily distinguished from other pseudo-oligo-saccharides by its biological, physical and chemical properties as set forth herein.

- Antibiotic 66-40 has a characteristic infrared absorption spectrum in mineral oil (Nujol) as shown by Figure i.

The more significant absorption bands are tabulated in Table D with the following designations: S=strong, M=medium, W=weak, brd=broad, VS=very strong and VW= very weak.

TABLE D Significant Infrared Absorption Bands of Antibiotic 66-40 2.98 u (M-S) 6.82 u (nujol) 10.46/u (M-S) 3.05/u (M-S) 7.25 i (nujol) 11.97 x (M) 3. I6 1 (M-S) 8.77/u (M-S) 12.75 /u (VW,brd.) 3. 35yu-3. 50 /u(nujol) 9.00/Ai (M-S) 13. 45-13- 90 i(W,brd) . 93 μ (W-M) 9. 7 (S) 6. 2 yu (M) 9.72-IO. O7 μ (S,brd.) Antibiotic 66-40 also has a characteristic nuclear magnetic resonance spectrum [NMR spectrum] as shown by Figure 2. The NMR spectrum was obtained by the use of a Varian A-60-A spectrometer (Varian Associates, 6ll Hansen Way, Palo Alto, California) on a solution (about 0. 4 ml; concentration about 20 mg/ml) of the antibiotic in deuterium oxide (DgO). The spectrum is recorded in parts per million (PPM) from 3-(trimethylsilo)-propane-sulfonic acid sodium salt, the internal standard.

In Table E is set additional data pertaining to qualitative, tests on and physical constants of Antibiotic 66-40 (free base).

TABLE , E a) Color Reactions Sakaguchi Starch-Potassium Iodide Ninhydrin Stannous Chloride Molisch Biuret b) Physical Constants Γ~ (C=0.3# in H O) + 188.9 Melting Point (Mdnohydrate) l85°-190°C Equivalent Weight 92 PKa 8.0 Elemental Analysis Found Calculated for a Monohydrate C 49.80 49.02 H 8. 20 8.44 N 14.95 15.04 0 (by difference) 27.05 27.50 Elemental Analysis corresponds to the formula C19¾7N5°7 , H2° Molecular weight as determined by mass spectrometry 447. 26 Ultra-violet absorption: transparent in the range between 220-400 mu. c) Solubility of Antibiotic 66-40 Base in Various Solvents Solvent Solubility* Methanol Sparingly soluble Acetone Insoluble - Solvent Solubility* Chloroform Slightly soluble Ether Insoluble Benzene Insoluble Water Very Soluble * Terminology is according to U.S. Pharmacopia XVIII, page 8.

In Table F is set forth the mass spectrum of Antibiotic 66-40 free base. In the table the columns headed m/e represents the mass to charge ratio, and the column headed Rel. Int. stand for Relative Intensity and as the name implies sets forth the intensities of the peaks at the various mass to charge ratios (m/e), relative to that of the peak m/e = ll8.

TABLE F Mass Spectrum of Antibiotic 66-40 Base- m/e Rel. Int. m/e Rel. Int. m/e Rel. Int. m/e Rel. Int. m/e R 40 3.0 75 3.0 113 11.0 146 11.0 199 2 41 30.0 80 24.0 114 14.0 ι4γ 3-5 200 2 42 63.Ο 81 19.5 115 2.5 151 2.0 201 3 43 .50.0 82 35.5 116 2.5 152 2.5 202 2 44 >100 83 I6.5 117 1.0 155 3.0 203 2 45 8.0 84 5I.O 118 100 (Base He) 16 2.0 205 6 46 5-0 85 21.0 119 8.0 157 2.5 206 6 51 2.5 86 58.Ο 120 2.0 158 4.5 215 6 52 3-5 87 28.Ο 121 2.0 159 3.5 216 2 53 10.0 88 11.0 122 2.0 l6o 98.Ο 219 2 54 10.0 92 7.0 123 2.0 161 9-5 220 2 55 20.0 93 6.0 124 3.0 162 2-0 236 7 56 50.0 94 I5.O 125 6.0 163 20.0 237 2 57 23.5 95 7.0 126 20.0 164 2.0 238 6 38 64.0 96 10.0 127 35.0 168 2.0 239 2 59 40.0 97 15.0 128 12.0 169 3-5 245 4 60 13-5 98 23.0 129 5.0 170 2.5 246 6 65 4.0 99 11.0 10 I5.O 173 19.O 253 2 66 3.5 100 44.0 131 2.5 174 3.0 254 4 67 12.0 101 14.0 133 2.0 176 3.0 255 2 68 65.5 102 17.0 135 2.0 178 7.0 256 1 69 21.0 107 3.9 138 2.0 185 2.0 270 2 70 I9.O IO8 7.5 140 3.0 186 2.5 271 1 71 30.0 109 I7.O 142 I6.O 187 I7.O 272 6 72 66.0 110 50.0 143 3.5 188 2.5 282 5 73 66.0 111 I3.O 144 6.0 191 32.0 289 4 74 28.Ο 112 I9.O 145 65.Ο 192 3.5 299 9 A, As mentioned before, this invention also relates to pharmaceutically acceptable derivatives of Antibiotic 66-40 , such as for example its solvates (e.g. hydrates), salts and condensation products with aldehydes and/or ketones. It is to be understood that the antibiotic can be subjected to more than one after-treatment; for example, solvates of salts of Antibiotic 66-40 are also comprised by the invention.

Some of these derivatives are described in detail below; Antibiotic 66-40 being basic readily forms non-toxic salts with organic and inorganic acids, such as for examp le hydrochloric, sulfuric, phosphoric, acetic, stearic, propionic, tartaric, maleic, benzoic acid and the like. Also partly neutralized (e.g. neutralized with an anorganic base such as, for example, NaOH or an organic base) polybasic acids can be used. In general, the mineral acid salts, such as those formed with hydrochloric acid, sulfuric acid, phosphoric acid and the like, are water soluble and may be obtained by concentration or lyophilization of an aqueous solution thereof or by precipitation with a water miscible organic solvent preferably a lower aliphatic alcohol or ketone. It is to be noted, however, that hydrochlorides of Antibiotic 66-40 exhibit substantial solubility in methanol and are, therefore, atypical. The hydrochlorides may be precipitated from an aqueous solution by the addition of a lower alkyl ketone, such as acetone. By titrating an aqueous solution of Antibiotic 66-40 with less than a stoichiometric amount of acid, it is possible to form partial acid addition salts. As used herein the term "acid addition salt" embraces all such compounds . - if The above-described non-toxic acid addition salts exhibit substantially the same antibiotic spectrum as does the free nitrogen base, however, they do differ in solubility characteristics.

The new antibiotic and its acid addition salts form hydrates with water and other solvates with organic sol vents. It becomes obvious, therefore, that in the isolation procedures described herein the antibiotic is normally obtained as a hydrate and its acid addition salts are normally obtained as solvates of lower aliphatic alcohols or ketones. These hydrates and solvates are relatively stable, therefore, the isolated products contain water or solvent, usually about one mole per mole of antibiotic.

Physical data of some derivatives of this sort are collected in Table G.

TABLE G Physical Constants of Antibiotic 66-40 Salts Antibiotic 66-40 Hydrochloride as Methanol Solvate [a]2^ (C=l# in H20) + 112.2° Elemental Analysis Found Calculated c 57.70 36.29 H 7.I5 7.00 N 10.61 IO.58 ci 25.60 26.78 Analysis corresponds to σηΛΗ,„Ν.-0„- 5HC1. CH,0H 19 3 5 7 3 Ultra-violet absorption: transparent in the range between 220-400 nu - e Antibiotic 66-40 Sulfate as Methanol Solvate [a]y(C«l# in H20) + 105.1° Elemental Analysis Pound Calculated C 32. 52 33.14 H 6.33 6.39 N 9 2 9.66 SO 33.90 3 . 13 Analysis corresponds to (C1QH_7N_0 ) .5H S0,.2CH,0H iy 3( 5 ( 2 £. *t j Ultra-violet absorption: transparent in the range bet-. ween 220-400 mju.

As mentioned before, Antibiotic 66-40 also forms nontoxic condensation products with aldehydes and/or ketones by procedures that are known in the art. The preferred products, probably Schiffbases, contemplated herein are those prepared by condensing the antibiotic with aldehydes and/or ketones having up to 12 carbon atoms. Included among such aldehydes and ketones are aliphatic, alicyclic, aromatic and heterocyclic compounds. Further, it is to be understood that the aldehydes and ketones having a closed ring structure may bear substituents such as hydroxy, halogeno, nitro, lower alkoxy, lower alka-noyloxy and the like. Solely for illustration and without limitation, the following are aoang the aldehydes and ketones contemplated herein: acetaldehyde, acetone, methyl-ethyl-ketone, crotonaldehyde, furfural, cyclo-pentylacetaldehyde, vanillin, veratraldehyde, benzo-phenone, benzaldehyde, acetophenone, salicylaldehyde, py-ridoxal and the like.

It is to be noted that these condensation products are not stable in the presence of water.

As mentioned before, Antibiotic 66-40 is produced when the elaborating microorganism, M. inyoensis is grown in an aqueous nutrient medium under aerobic, preferably submerged conditions.

For limited amounts of antibiotic, surface culture in bottles or shake flasks can be employed instead of submerged conditions. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate. An assimilable nitrogen compound or proteinaceous material is also required. Preferred carbon sources include glucose, maltose, mannose, sucrose, starch, corn. starch and the like. Preferred nitrogen sources include corn steep liquor, yeast extracts, soybean meal, meat peptones, casein hydrolysates, beef extracts and the like. Combinations of these carbon and nitrogen sources can be used advantageously. It generally is not necessary to add trace elements since tap water is used as the formulation medium; however, addition of cobalt salts has been found to be advantageous.

Production of Antibiotic 66-40 can be effected at any temperature conducive to satisfactory growth of the microorganism, for example between 20° and 0°C, pre- ■ ferably 25-35°C Ordinarily optimum production is obtained in' 3-7 days. The pH of the medium usually stays fairly close to 7 during the fermentation. The final^pH is in part dependent on the buffers present, if any, and is advantageously adjusted to about 8.0 prior to sterilization.

When growth is carried out in large vessels and tanks, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the broth culture with a soil or slant culture or a lyophilized culture of the - organism. When an active inoculum has been so obtained, it is transferred aseptically to larger vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as or different from that utilized for the production of the antibiotic in tanks as long as good growth of the microorganism is obtained.

After completion of the fermentation, the medium may, for instance, be worked up as follows: The whole broth is adjusted to a pH of about 2 with mineral acid, preferably aqueous sulfuric acid, whereby the basic water-soluble antibiotic is released from the mycelium and dissolved in the aqueous fermentation medium. The entire mixture is filtered so as to remove the broth, and the filtrate is neutralized, followed by addition of oxalic acid to precipitate calcium ions. After an additional filtration and adjustment to neutrality, preferably with ammonium hydroxide, the clear neutralized filtrate is passed through an ion exchange resin, preferably of the IRC-50 Amberlite type in the ammonium form.

Examples of the Amberlite type resins employed herein, both for anionic and cationic exchange, are found in the Handbook of Chemistry and Physics, 42nd Edition, Chemical Rubber Publishing Company, Cleveland, Ohio (i960). The spent broth is discarded, and the antibiotic is eluted /from the resin with ammonium hydroxide. The eluate is concentrated and evaporated to a residue consisting of crude Antibiotic 66-40 having a potency of about 500 mcg/mg according to the assay procedure hereinafter described.

Purification of the crude antibiotic may be effected by utilization of an anionic exchange resin, preferably Dowex 1 X 2 or an Amberlite IRA 401S type. The crude antibiotic may be adsorbed from an aqueous solution onto the column and eluted therefrom with distilled water. Material obtained in this manner assays approximately 900 mcg/mg. Alternatively, for instance, the crude antibiotic is purifiable by column chromatography on cellulose utilizing a solvent system of chloroform: methanol; 17 ammonium hydroxide (2:1:1); the upper phase of the solvent system is used to first "wet" the column, and the lower phase is used for elution purposes. The antibiotic may be placed on the column by adsorption from a concentrated solution in the upper phase of the aforementioned solvent system.

In order to assay the various preparations for potency in terms of micrograms .per milligrams of the antibiotic, the standard cylinder cup assay method is usually employed using Staphylococcus aureus ATCC 6538P (ATCC = American Type Culture Collection) as test organism.

This method is completely analogous to that described by Oden et al. in Antimicrobial Agents and Chemotherapy (1963): One microgram of antibiotic activity of the antibiotic is the amount of material which produces a zonal response of l6, 3 - 0,9 mm under the conditions of the assay method and is expresses as mcg/mg.

The following examples are illustrative of fermentation and isolation procedures yielding Antibiotic 66-40: EXAMPLE 1 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 3OO 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 1000 ml Incubate the flask and its contents for five days at °C on a rotary shaker ( 280 rotations per minute, [r.p.m], 50, 8 mm stroke).

Germination Stage 2 ; Aseptically transfer 25 ml of the fermentation medium of germination stage 1 to a two-liter shake flask containing 500 ml of the aforedescribed sterile germination medium. Incubate the flask and its contents for three days at 28°C on a rotary shaker ( 280 r.p.m., 50 , 8 mm stroke).

Fermentation stage; Aseptically transfer 500 ml of the medium obtained from germination stage 2 to a 14-liters fermentation tank containing 9, 5 liters of the following sterile medium: Dextrin 50 g Dextrose 5 g Soybean me 35 g Calcium carbonate 7 g Cobalt chloride 10 molar Tap water 1000 ml Antifoam (GE60=sili- Ίθ ml cone emulsion) Prior to sterilizing the aforedescribed mediium,adJust the pH to 8. Aerobically ferment for 66-90 hours while stirring at 250 r.p.m. with air input at 4,5 liters per liter/minute and 22,5 at. The potency of the antibiotic produced at the end of this period reaches a peak of 150-225 mcg/ml and remains relatively constant. The pH of the fermentation medium changes slightly during the antibiotic production, varying in the range of 6,8 - 7,3.

EXAMPLE 2 ϊ5° l of_Antibiotic_66-40 The whole broth from Example 1 is adjusted to pH 2 with 6N sulfuric acid. (For the purpose of this example, quantities are given in terms of 170 liters of fermentation broth obtained by pooling acidified broths from 17 batches obtained according, to the procedure of Example 1). 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 15OO-2000 Amberlite in its ammonium form. Discard the . θϋ^έβ^ wash the resin with water, and elute with 2N ammonium hydroxide. Collect 400 ml fractions and' monitor by disc testing with S. aureus AT'.QC 6538P.

Combine active fractions and evaporate to dryness under vacuum obtaining about 28 g of crude Antibiotic 66-40 having an activity of about 500 mcg/g.

EXAMPLE 3 Purification of Antibiotic 66-40 Dissolve 28 g of crude Antibiotic 66-40 obtained in Example 2 in 100 ml of distilled water and charge to an anion exchange adsorption column (Dowex 1X2) in the hydroxyl form. Slurry 2000 g of the resin in water into a column 63,5 mm in diameter and 914,4 mm high. Elute the column with distilled water at a rate of about 2> 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: Y 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 mcg/mg.

EXAMPLE 4 Preparation of Antibiotic 66-40 sulfate Dissolve 3·9 g of Antibiotic 66-40 base prepared as described in Example 3 in 60 ml of water and adjust the pH to 4,5 with 6N sulfuric acid. Stir the solution with decolorizing charcoal for about 1/2 hour and filter. Add the filtrate to about 1 liter of methanol. Filter and dry obtaining 4,8 g of the sulfate salt; assay about 640 mcg/mg.

EXAMPLE 5 Alternate Purification of Antibiotic 66-40 via its Sulfate 200 g of Whatman No. 1 cellulose powder are mixed with 20 ml of the top phase of a solvent system composed of chloroform: methanol: 17 ammonium hydroxide (2:1:1) and packed in small segments in a column having an inner diameter of 6, 35 mm and a height of 508 mm. The lower phase of the solvent system is run through the column until a yellow band of impurities emerges. Two grams of Antibiotic 66-40 sulfate prepared as described in Example 4 a e dissolved in about 3 ml of the upper solvent phase, mixed with some cellulose powder, dried under vacuum and packed atop the cellulose column. Lower phase is allowed to run through the column at the rate of 1 ml per minute collecting 5 ml fractions every 15 minutes.

Aliquots of each fraction were spotted on filter paper and tested with ninhydrin reagent to determine the presence or absence of antibiotic. Paper chromatography of the antibiotic-containing fractions established that the desired material was located between fractionsl21 to 190.

Fractions 121 to 190 are combined, evaporated to dryness, rediasolved in water, and passed through IRA 401S (an anion exchange resin) in the hydroxyl cycle. he pH of the eluate the eluate is adjusted to 4, 5 with sulfuric acid;xni∑t treated with charcoal, filtered and concentrated to a smaller volume. The concentrate is added to an excess amount of methanol and the white precipitate that is formed is separated by filtration. The precipitate is dissolved in water and passed through an IRA 401S resin column in the hydroxyl form. The effluent is collected, concentrated and lyophilized, yielding about 300 mg of Antibiotic 66-40 IK-Je EXAMPLE 6 Preparation of Antibiotic 66-40 Hydrochloride Dissolve 104,7 mg of Antibiotic 66-40 base, prepared as described in Example 2 in 4 ml of water and adjust the pH to 4,5 with hydrochloric acid. Evaporate the solution to dryness and redissolve the residue in methanol. Add this solution to excess acetone and filter the resulting precipitate obtaining 1^0 mg of Antibiotic 66-40 hydrochloride; assaying at 753 mcg/mg.

EXAMPLE 7 Preparation of Crystalline Antibiotic 66-40 Monohydrate Prepare a (26 x 2,5 cm) silica gel chromatographic column using the lower (organic) phase of a solvent mixture consisting of isopropanol: CHCl^: ammonium hydroxide in the volume ration of 1:2:1 as the developer/eluent . Dissolve 1,0 gram of Antibiotic 66-40 base in 5,0 ml of solvent mixture. Adsorb the antibiotic solution to the silica gel and chromatograph. Collect *0 ml fractions and determine the location of the desired fractions by thin-layer chromatography on silica gel plates. Combine and evaporate the appropriate (44-78) fractions in vacuo and obtain thereby a pale yellow syrup which upon azeotropic distillation with ethanol crystallizes as pale yellow rosettes. The product obtained in this manner is Antibiotic 66-40 monohydrate which melts at about l85-190°C. The yield is about 6 0 mg.

IK-je EXAMPLE 8 Preparation of Benzaldehyde Condensation Product of An^ibiotic_66-40 ,0 g of Antibiotic 66-40 in 60 ml absolute ethanol is treated with 5,9 g benzaldehyde (slight excess over equivalents) and refluxed for 1 hour. The solution is cooled and filtered affording 7 ,0 g of a white crystalli-ne solid, m.p. 12 126°C. [a] ° = +4J.20 (C = 0,J>% in CHCl-j). Elemental analysis indicates five benzaldehyde residues.

Similarly, by replacing the benzaldehyde reactant in the foregoing example with an equivalent quantity of any one of the following aldehydes acetaldehyde, furfural, eyelopentylacetaldehyde, crotonaldehyde, salicylaldehyde, vanillin, veratraldehyde or pyridoxal and by following substantially the procedure set forth in the example, the corresponding aldehyde condensation products (probably Schiff's Bases in terms of chemical structure) are obtained.

Antibiotic 66-40 and its pharmaceutically acceptable functional derivatives possess a broad antibacterial spectrum. The antibiotic has the property of adversely affecting the growth of gram positive and gram negative bacteria and thus can be used alone or in combination - e ι with other antibiotic agents to prevent the growth of or reduce the number of bacteria in various environments.

It may be used, for example, to disinfect laboratory glase-ware, dental and medical equipment contaminated with Staphylococcus aureus or other bacteria whose growth is adversely affected by Antibiotic 66-40. Because of its particularly effective activity against gram negative bacteria, it is useful in combatting infections caused by such gram negative organisms.

The in vitro activity of Antibiotic 66-40 against a variety of gram positive and gram negative bacteria is set forth in Table H. The minimal inhibitory concentration (MIC) was determined utilizing yeast beef broth as the te:at medium. A two-fold serial dilution technique was employed. The MIC is the mid-point between the last clear tube and the first turbid tube determined by visual observation. Determinations were made using a ~ dilution of a 2 hour broth culture of the test bacteria. All tubes were incubated for l8 hours at j57°C.

In the table, Antibiotic 66-40 having a potency of 1000 mcg/mg was used for the study.

TABLE H In Vitro Activity of Antibiotic 66-40 Microorganisms Used MIC Gram Positive Bacteria mcg/ml Diplococcus pneumoniae DA 150 Enterococcus sp. DA 800 Enterococcus sp. DA 801 Enterococcus sp. DA 802 Staphylococcus aureus ATCC 6 38P Staphylococcus aureus ATCC II63I - e Staphylocpccus aureus Gray 0.05 Staphylococcus aureus DA 2033 0.08 Streptococcus faecalis ATCC 10541 3.0 Streptococcus pyogenes DA 1 3.0 Streptococcus pyogenes DA 21 3· 7 Streptococcus pyogenes DA 15 3·7 Gram Negative Bacteria Escherichia coli ATCC 10 30 0.6 Escherichia coli DA 0.3 Escherichia coli DA 4 0.6 Escherichia coli DA 1 0.6 Klebsiella pneumoniae DA 20 0.23 Klebsiella pneumoniae ATCC 10031 3.0 Proteus vuHgaris DA 121 0.6 Proteus vulgaris ATCC 9921 0.6 Proteus vulgaris DA 13 0.3 Proteus vulgaris DA 12 3.0 Pseudomonas aeruginosa ATCC 8709 0.45 Pseudomonas aeruginosa ATCC 8689 0.6 Pseudomonas aeruginosa ATCC 9027 0.6 Salmonella schottmauelleri DA 10 0.53 Salmonella sp.. DA 101 0.3 Salmonella sp. DA 102 0.3 Aerobacter sp. DA 3a 0.3 -■■DA refers to Schering Corporation collection number.

The acute toxicity of Antibiotic 66-40 in the form of its sulfate was determined in the standard manner by variety of routes in mice weighing 18-20 grams. The toxicity data given in Table J is expressed in terms of the free base.

I - e TABLE J Acute Toxicity of Antibiotic 66-40 Mode of Administration LD.r0(mgAg ) subcutaneous 288 intraperitoneal 221 intravenous j5 Antibiotic 66-40 exhibits an antibacterial action against pathogenic bacterial infections induced in laboratory animals and .in particular in the mouse. To determine the in vivo protective activity of Antibiotic 66-40 against infections of pathogenic bacterial origin in mice, mice were dosed twice with the antibiotic, once immediately before an intraperitoneal injection of the infecting bacte ria and once 4 hours after such injection. The number of survivors was determined 48 hours after infection and that data analyzed by standard probit procedures to determine PD 5-.0- values with 95$ confidence limits. Table K sets forth the protective activity of Antibiotic 66-40 against various pathogenic bacteria.

TABLE K Protective Activity of Antibiotic 66-40 in Mice Route of Organism Administration ¾o- Staphylococcus aureus Gray DC 445 subcutaneous 0 , 12 oral 25, 0 Streptococcus pyogenes C DC 28 subcutaneous 0, 87 Klebsiella pneumoniae DC 801 subcutaneous 0, 70 oral 50, 0 Pseudomonas aeruginosa ATCC 8709 subcutaneous 1, 12 Salmonella paratyphi B DC 837 subcutaneous 1, 91 - e It is ■ evident from Table K that the therapeutic index (LD-.0/PD,-0 ) via the subcutaneous route ranges from 5^0 to 2400 with regard to the gram positive organisms and 150 to 410 with regard to the gram negative organisms.

In addition, mice infected intraperitoneally with eight LD,-,, doses of Rickettsia akaria were afforded 100$ protection by the subcutaneous administration of 2 mg of Antibiotic 66-40 administered once a day for four days.

In view of the foregoing in vivo data and especially in view of the favorable therapeutic index exhibited by Antibiotic 66-40, it is evident that the antibiotic may be used to control and treat a variety of infections in mammalian hosts. Among such infections are those caused genug by 3&&.«:¾&3- of such organisms as Streptococcus, Staphylococcus, Streptococcus, Escherichia, Salmonella, Klebsiella, Proteus, Pseudomonas and the like. The foregoing organisms cause or are suspected of causing bovine mastitis, urinary tract infections and diarrhea. Species of the same organisms are suspected of causing skin and upper respiratory diseases or of aggravating pre-existing manifestations of such diseases in mammals. Antibiotic 66-40 and its derivatives, therefore, provide a potent weapon for combatting such organisms and disease states caused thereby.

Antibiotic 66-40 and its derivatives may be applied topically in the form of ointments, both hydrophilic and hydrophobic, in the form of lotions which may be aqueous,' nonaqueous or of the emulsion type or in the form of creams. Pharmaceutical carriers useful in the preparation of such formulations will include, for example, such substances as water, oils, greases, polyesters, polyols and the like.

- In general, the topical preparations will contain from about 0, 1 to about 3, 0 g of the antibiotic per 100 g of ointment, cream or lotion. The topical preparations are usually applied gently to lesions from about 2 to about 5 times a day.

The antibiotics of the instant invention may be utilized in liquid form such as solutions, suspension and the like for otic and optic use and may also be administered parenterally via intramuscular injection. The injectable solution or suspension will usually be administered at from about 1 mgto about 5 mg of antibiotic per kilogram of body weight per day divided into about 2 to about 4 doses. The precise dose depends on the stage and severity of the infection, the susceptibility of the infecting organism to the antibiotic and the individual characteri-sties of the being treated.

Example 9 below sets forth the ingredients and the process for making an injectable solution.

EXAMPLE 9 Injectable_Solution Per 2.0 ml Vial Per 50 liters # Antibiotic 66-40 sulfate 84,0 mg 2100, 0 g p-Hydroxyb'enzoic acid 3,6 mg 90, 0 g methyl ester, USP (= ethylparaten) p-Hydroxybenzoic acid propylparaben0' mg 10, 0 g propyl ester, USP7^ (=f½t¾^¾3¾ra]-m J Sodium bisulfite, USP 6,4 mg 160 g Disodium Ethylenediamine- 0 2 mg 5, 0 g tetraacetate Dihydrate, R.G.

Water for injection, USP q.s. ad 2,0 ml 50, 0 liters * Includes a 5% manufacturing overcharge US-Pharmacopoea Procedure: For a 50^0 liter batch Charge approximately 35 liters of water for injection to a suitable stainless steel jacketed vessel and heat to about 70°C. Charge the methylparaben and propylparaben to the heated water for injection and dissolve with agitation. When .the parabens are completely dissolved, cool the contents of the tank to 25-J>0°C by circulating cold water through the tank jacket. Sparge the solution with nitrogen gas for at least 10 minutes and keep covered with nitrogen during subsequent processing. Charge and dissolve the disodium ethylene diamine-tetra acetate dihydrate and sodium bisulfite. Charge and dissolve the Antibiotic 66-40 sulfate. Bring the batch volume up to .50,0 liters with water for injection and agitate until homogenous.

Under sterile conditions, filter the solution through a suitable bacteria retentive filter collecting the filtrate in a filling tank.

Fill the producc aseptically into sterile pyrogen-free multiple dose vials, stopper and seal.

EXAMPLE 10 Antibiotic Ointment Antibiotic 66-40 base 10 g Petrolatum 990 g Procedure (1) Melt the petrolatum; (2) Admix Antibiotic 66-40 base with about 10$ of the molten petrolatum; - '(3) Pass the antibiotic - petrolatum mixture through a colloid mill; 4) Add in the remainder of the petrolatum and cool the mixture until it becomes semi-solid. At this stage the product may be put into suitable containers.

Claims (29)

April- -1972 951 A Israel ED-MK WHAT WE CLAIM IS:

1. Antibiotic 66-40 having in its 'free form a gross structural formula substantially as follows: and being (a) in its free form or (b) in the form of a pharmaceutically acceptable acid addition salt or (c) in the form of a pharmaceutically acceptable condensation product with an aldehyde or mixture of aldehydes or (d) in the form of a pharmaceutically acceptable solvate of (a), (b) or (c) .

2. A product comprising Antibiotic 66-40 having in its . free form an infrared absorption spectrum in mineral oil substantially as shown in Figure 1 and a nuclear magnetic spectrum in deuterium oxide substantially as shown in Figure 2, and physical data as given in Tables E and F herein, the Antibiotic being (a) in its free form or (b) in the form of a pharmaceutically acceptable acid addition salt or (c) in the form of a pharmaceutically acceptable condensation product with an aldehyde or (d) in the form of a pharmaceutically acceptable solvate of (a) or (b) .

3. A product comprising Antibiotic 66-40 having in its free form an infrared absorption spectrum in mineral oil substantially as shown in Figure 1 and a nuclear magnetic spectrum in deuterium oxide substantially as shown in Figure 2, and physical data as given in Tables E and F herein, the Antibiotic being in the form of a pharmaceutically acceptable condensation product with a mixture of aldehydes or in the form of a pharmaceutically acceptable solvate of a pharmaceutically acceptable condensation product with an aldehyde or mixture of aldehydes.

4. A product according to claim 2, having in its free form a gross structural formula substantially as follows

5. A solvate according to any preceding claim.

6. A product according to claim 5> wherein the solvate is a hydrate.

7. A product according to claim 6, said product being Antibiotic 66-40 monohydrate in substantially pure crystalline form.

8. A product according to any one of claims 1 to 6, said product being an acid addition salt.

9. -A product according to claim 8, said product bein an acid addition salt, which when derived from a polybasic acid may contain free acid groups and/or acid groups neutralized by other bases.

10. A product according to claim 9, containing acid moieties of at least one of the following acids: hydrochlori sulfuric, phosphoric, acetic, stearic, propionic, tar- taric, maleic and benzoic acid.

11. A product according to any one of claims 1 to 6, said product being a condensation product with an aldehyde or mixture of aldehydes.

12. A product according to claim 11, wherein the aldehyde moiety has up to 12 carbon atoms.

13. 5. A product according to claim 12, wherein the aldehyde is benzaldehyde .

14. Antibiotic ββ- θ in any of forms (a), (b), (c) and (d) specified in claim 1 and substantially as hereinbefore described.

15. Process for the production of an antibiotically active product comprisin Antibiotic ββ- θ, in any of forms (a), (b), (c) and (d) specified in claim 1 which comprises incubating a microorganism of the. species Micro- monospora inyoensis in an aqueous nutrient medium containing assimilable sources of at least nitrogen and carbon under aerobic conditions until substantial antibacterial activity is imparted to said medium and iso- lating the said antibiotically active product the e)^

16. Process according to claim 15, wherein the incubation is performed under submerged conditions.

17. Process according to claim 15 or 16, wherein the microorganism incubated is Micromonospora inyoensis NRRL 3292 or an Antibiotic 66-40 producing mutant or variant thereof, possessing. substantially the same biological activity.

18. Process according to any one . of claims 15 to 17., wherein the microorganism is incubated at a temperature from about 20 to o°C, and at a pH from 7 to 8.

19. Process according to claim l8. where the microorganism is incubated at a temperature from 25 to 35°C.

20. Process according to any one of claims 15 to 19^ wherein the antibiotically active product is recovered by: freeing the antibiotically active product from the mycelium, adsorbing the antibiotically active product on an ion-exchange resin, washing the resin adsorbate with water, eluting the antibiotically active product from the resin, concentrating the.eluate, and isolating the antibiotically active product from the concentrate.

21. Process according to any one or claims 15 to 20,, wherein chromatographic methods are used for the purification of the antibiotically active product.

22. Process according to any one of claims 15 to 21, which includes at least one of the following after-treatments, carried out after formation of the antibiotically active product: a) recipitation as a solvate out of an organic or aqueous solution by adding a solvent wherein the antibiotically active product is not or onl slightly soluble; (b) addition of an acid or acid derivative so as to obtain an acid addition salt, which when derived from a polybasic acid may contain free acid groups and/or acid goups neutralized by other bases; (c) condensation with an aldehyde or mixture of aldehydes.

23. Process according to claim 22 in which the or each aldehyde has up to 12 carbon atoms.

24. Process for the production of an antibiotically active product comprising Antibiotic 66-40, in any of forms (a),, (b), (c) and (d) specified in claim 1, substantially as -described herein.

25. . Antibiotic 66-40 in any of forms (a), (b), (c) and (d) when obtained by the process of any one of claims 15 to 24.

26. Method for the preparation of a therapeutical composition having antibiotic activity, characterized in that an antibiotically active product comprising Antibiotic 66-40 in any of forms (a), (b), (c) and (d) specified in claim 1, as an active ingredient is brought into a form suitable for therapeutical administation.

27. . A method according to claim 26 which comprises mixing the antibiotically active product v/ith a suitable pharmaceutical carrier.

28. Method for the preparation of a therapeutical composition according to claim 26 and substantally as hereinbefore described.

29. Antibiotically active composition, characterized : by a antimicroblally effective content of an antibiotically active product comprising Antibiotic ββ- θ in any of forms (a), (b), (c) and (d) specified in claim 1, as an active ingredient in admixture with a suitable pharmaceutical carrier.

0. Composition according to claim 29, characterized in that it is a shaped composition of matter, such as a tablet or capsule. ·