Abstract:
The novel antibiotic, lipiarmycin, is prepared by cultivating Actinoplanes deccanensis A/10655, ATCC 21983, under aerobic conditions in a fermentation broth, from which it is then recovered.

Description:
SUMMARY OF THE INVENTION 
     In the preparation of the novel antibiotic lipiarmycin, Actinoplanes deccanensis A/10655, which has been deposited and made part of the stock culture collection of ATCC, where it was assigned number 21983, is cultivated under aerobic conditions in an aqueous nutrient medium suitable for the growth of said organism, the medium containing a source of carbon, a source of nitrogen and inorganic salts. Ordinarily, the antibiotic-producing strain is pre-cultured in a shake flask until substantial antibiotic activity is present, then the culture is used to inoculate jar fermentors containing nutrient fermentative medium. Cultivation is continued at 25°-35°C under aerobic conditions for a time sufficient to produce a substantial antibiotic level. During this time, microbiological assays are carried out by the agar diffusion method to control the concentration of the antibiotic substance produced. The lipiarmycin is isolated from the fermentation broth by conventional procedures, such as, for instance, by extraction with an organic solvent in which the antibiotic substance is soluble and which is immiscible with the aqueous medium. Suitable organic solvents for such purpose are advantageously selected from halogenated C 1  -C 4  hydrocarbons and C 4  -C 6  alkanols. The solvent is then separated from the fermentation broth by high-speed centrifugation, concentrated to about 1/20-1/40 of its original volume and allowed to stand until the antibiotic substance precipitates. 
     The crude lipiarmycin is dissolved in a 90:10 chloroform:methanol mixture, chromatographed through a silica gel column and eluted with a mixture of chloroform and methanol in the same proportions as above. Lipiarmycin is finally crystallized from a mixture of diethyl ether and light petroleum. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Production of Lipiarmycin 
     To produce the antibiotic lipiarmycin, the strain Actinoplanes deccanensis A/10655 is aerobically pre-cultured in a nutrient medium until substantial antibiotic activity is present at a pH value ranging from about 6 to about 8. As an example, a shake flask culture may have the following composition in g./l. 
     
         ______________________________________Meat extract           3.0Tryptone               5.0Yeast extract          5.0Glucose                1.0Soluble starch         24.0Calcium malate         4.0Distilled water q.s. to                  1000 ml.______________________________________ 
    
     The flasks are shaken for about 24 hours at about 28°-30°C., then the pre-cultures (one liter) are used to inoculate jar fermentors, each containing 10 liters of the following nutrient medium: 
     
         ______________________________________Meat extract           40 g.Peptone                40 g.Yeast extract          10 g.Sodium chloride        25 g.Soybean meal           100 g.Glucose                500 g.Calcium carbonate      50 g.Tap water q.s. to        10 liters______________________________________ 
    
     The fermentation batches are incubated aerobically under stirring at 28°-30°C. At intervals, the antibiotic activity is assayed microbiologically by the agar diffusion method using Staphylococcus aureus as the test organism. The maximum activity is reached after 72- 96 hours of fermentation. 
     Isolation of Lipiarmycin 
     When fermentation is completed the fermentation broth is extracted twice with an amount of butanol corresponding to 30 percent of its volume. The butanol solution is separated from the broth by high-speed centrifugation and is concentrated to about 1/20 of its original volume by evaporation under vacuum at 40°-50°C. The butanol solution so obtained is washed with water and the resulting two layers are separated. The organic layer is further concentrated to about 1/30 of its original volume and allowed to stand for about 3-7 hours at 4°C. until a precipitate forms, which is recovered by filtration. By addition of light petroleum to the filtrate, further crude compound is obtained. From 10 liters of fermentation broth, 11 g. of antibiotic substance are recovered. 
     The so obtained crude lipiarmycin is dissolved in a 90:10 chloroform:methanol mixture and to the resulting solution a compatible amount of silica gel is added. The mixture is evaporated to dryness under vacuum at 40°-50°C., the solid obtained is added to the top of a silica gel column and the subsequent elution is carried out with a mixture of chloroform and methanol in the same proportions as above. 
     The so obtained purified antibiotic is dissolved in a small amount of methanol and to the resulting solution is added diethyl ether and the resulting solution is heated to about 40°C. for a few minutes. Light petroleum is added to this warm solution until a slight opacity is observed, then the whole is allowed to stand at 3°-6°C. for one day. Pure lipiarmycin thereby precipitates, which is recovered by filtration and dried under vacuum. 
     Lipiarmycin as so produced is a white crystalline substance having the following properties: 
     1. Melting point: 173°-75°C. (from a mixture of methanol, diethyl ether and light petroleum) 
     2. Molecular weight: 1076 (potentiometric determination) 
     3. Elemental analysis: C 58.02%; H 6.94%; Cl 6.64%; O 28.40% (by difference) 
     4. U.V. absorption bands 
     In each of the below-outlined solvent systems, lipiarmycin shows the following values: 
     
         ______________________________________Solvent       max (m μ)    E.sub.1 cm..sup.1%______________________________________methanol      232             354         268 (shoulder)  214         315             108phosphatebuffer pH 7.38         238             331         275             194hydrochloricacid 0.1 N    231             338         272             207sodium hydroxide0.1 N         235             370         270 (shoulder)  183______________________________________ 
    
     The complete picture of the spectrum is given in accompanying FIG. 1. 
     5. Infrared Spectrum 
     Characteristic absorption bands occur at the following frequencies (cm.  -   1 ): 3600, 3450, 2900(Nujol), 1730, 1690, 1640, 1585, 1560, 1460(Nujol), 1380(Nujol), 1300, 1240, 1200, 1140, 1120, 1075, 1025, 990, 950, 915, 900, 890, 850, 820, 790, 780, 745, 720, 705. 
     A complete figure of the I.R. spectrum is given in accompanying FIG. 2. 
     6. Specific rotation: [α ]   D   20  (1.98 percent methanol) = -5.5° 
     7. Proton Magnetic Resonance (P.M.R.) spectrum: see accompanying FIG. 3. 
     8. Solubility 
     Very soluble in ethanol, methanol, pyridine, aqueous sodium carbonate. 
     Fairly soluble in benzene, methylene chloride, chloroform, acetone, propanol. 
     Sparingly soluble to insoluble in water, buffered solution at pH 7, aqueous sodium bicarbonate, hexane. 
     9. Characteristic reactions 
     
         ______________________________________Tollens          positiveFeCl.sub.3       positiveH.sub.2 SO.sub.4 positiveSchiff           negativeMillon           negativeMaltole          negative______________________________________ 
    
     10. Acidity 
     An ionizable function is spectrophotometrically evidenced with pKa (in methyl cellosolve): 6.8 
     On the basis of microanalytical data, lipiarmycin may be assigned the following empirical formula: C 52  H 74  Cl 2  O 19 . 
     The antibiotic substance of the invention is active against bacteria, and in particular against the following microorganisms at the indicated concentrations. 
     
         ______________________________________                 MINIMAL                 INHIBITORY                 CONCENTRATIONSTRAIN                γ/ml.______________________________________Staphylococcus aureus ATCC 6538                  2Staphylococcus aureus Tour                  2Streptococcus hemolyticus C203                 10Diplococcus pneumoniae UC 41                 50Staphylococcus aureus Tour with 10                 10percent bovine serumStreptococcus mutans ATCC 25175                 0.5Mycobacterium tub. H37Rv ATCC 9360                 50Mycoplasma gallisepticum H 21 C.Z.B.                 50______________________________________ 
    
     Lipiarmycin is also active against strains which are resistant to other antibiotics which are widely used in chemotherapeutic practice. As a representative example, in the following table the minimum inhibitory concentrations (M.I.C.), γ/ml., of lipiarmycin against Staphylococcus aureus strains resistant to several antibiotics are reported. 
     
                       Table II______________________________________          M.I.C.         M.I.C.          of other       ofSTRAIN        antibiotics     lipiarmycin______________________________________Staphylococcus aureusATCC 6538 resistantto penicillin penicillin &gt;100 2Staphylococcus aureusATCC 6538 resistantto streptomycin         streptomycin &gt;100                         5Staphylococcus aureusATCC 6538 resistantto tetracycline         tetracycline &gt;100                         5Staphylococcus aureusATCC 6538 resistantto novobiocin novobiocin &gt;100 2Staphylococcus aureusATCC 6538 resistantto neomycin   neomycin &gt;100   2Staphylococcus aureusATCC 6538 resistantto erythromycin         erythromycin &gt;100                         5Staphylococcus aureusATCC 6538 resistantto chloramphenicol         chloramphenicol &gt;100                         2Staphylococcus aureusATCC 6538 resistantto cephaloridine         cephaloridine &gt;100                         5Staphylococcus aureusATCC 6538 resistantto streptothricin         streptothricin &gt;100                         5Staphylococcus aureusATCC 6538 resistantto bacitracin bacitracin &gt;100 5Staphylococcus aureusATCC 6538 resistantto oleandomycin         oleandomycin 50 5______________________________________ 
    
     These favorable antimicrobial properties are coupled with a very low toxicity, the LD 50  value of lipiarmycin being about 500 mg./kg. i.p. in mice. Accordingly, the present invention provides a therapeutic composition comprising the compound of the invention together with a pharmaceutically-acceptable carrier. 
     Description of Actinoplanes deccanensis A/10655 
     This strain grows well on many agars. The surface is opaque and slightly rough to wrinkled. Aerial mycelium is always absent. At microscopic examination the vegetative mycelium is branched, with a diameter of about 1 μ. The sporangia form abundantly on soil extract agar and are globose with an irregular surface and a diameter ranging from 4-7 μ. After rupture of the wall of the sporangium, it is possible to observe spore release. The spores are sub-spherical and are motile (size 1 μ × 1.5 μ). 
     Table III reports the cultural characteristics of Actinoplanes deccanensis A/10655 when cultivated on various standard media suggested by Shirling and Gottlieb (Intern. J. Syst. Bact., 16, 313-340, 1966) and other media recommended by Waksman (The Actinomycetes, vol. II, The Williams and Wilkins Co., 1961). The cultural characteristics were determined after 6 to 14 days of incubation at 30°C. 
     
                       Table III______________________________________The numbers of some of the culture media refer tothose given by Shirling and Gottlieb.Culture Media    Cultural Characteristics______________________________________Medium No. 2 (yeast            Abundant growth, veryextract-malt agar)            wrinkled, light orangeMedium No. 3 (Oatmeal            Moderate growth, crusty,agar)            light amberMedium No. 4 (Inorganic            Abundant growth, crusty,salts-starch agar)            orangeMedium No. 5 (Glycerol-            Moderate growth, roughasparagine agar) surface, orangeMedium No. 6 (Peptone-            Moderate growth, wrin-yeast extract-iron agar)            kled, light orangeMedium No. 7 (Tyrosine            Abundant growth, wrin-agar)            kled, amber to light            brown, diffusible brown,            pigmentOatmeal agar according            Abundant growth, crusty,to Waksman       opaque, light orangeHickey and Tresner&#39;s            Abundant growth, wrin-agar             kled, light orange-            pinkishCzapeck glucose agar            Moderate growth, crusty,            cream to light orangeGlucose asparagine            Moderate growth, slight-agar             ly crusty, opaque, cream            to light orangeNutrient agar    Moderate growth, crusty,            opaque, light orangePotato agar      Abundant growth, very            wrinkled, pale orangeBennett&#39;s agar   Abundant growth, wrin-            kled, light orangeCalcium malate agar            Scanty growth, wrinkled,            opaque light orangeSkim milk agar   Abundant growth, wrinkled,            opaque orangeCzapeck agar     Moderate growth, crusty,            light orangeEgg agar         Scanty growth, thin,            opaque, white-waxyPeptone glucose agar            Moderate growth, crusty,            orangeAgar             Very scant growth, thin,            hyalineLoeffler serum   Moderate growth, rough            surface, orangePotato           Moderate growth, wrinkled,            light orangeGelatin          Scanty growth, light            orangeCellulose agar   Very scanty growth, thin,            hyaline______________________________________ 
    
     The most convenient temperature for development of the colonies was found to range from about 18° to about 42°C.; the optimum temperature being from about 28° to about 37°C. 
     Table IV reports the utilization of carbon sources examined according to the method of Pridham and Gottlieb. 
     
                       Table IV______________________________________Carbon sources    Utilization______________________________________Inositol          -Fructose          -Rhamnose          +Mannitol          -Xylose            +Raffinose         -Arabinose         +Cellulose         -Sucrose           +Glucose           +Mannose           +Lactose           +Salicin           -______________________________________ 
    
     Following Table V reports the physiological characteristics of the strain. 
     
                       Table V______________________________________TEST                  RESULTS______________________________________Hydrolysis of starch  positiveH.sub.2 S formation   negativeTyrosinase reaction   positiveCasein hydrolysis     negativeSolubilization of calcium malate                 positiveNitrate reduction     positiveLiquefaction of gelatine                 positive       coagulation   negativeLitmus milk       peptonization negativeCellulose decomposition                 negativeChromogenic action    positive______________________________________