BE697319A - - Google Patents

Description

  

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  New antibiotics.



   The present invention relates to a novel antibiotic substance and its preparation.



   The subject of the invention is an antibiotic substance comprising a complex of tenebrimycins or the antibiotics distinct from this complex, from which mixtures of these distinct antibiotics or of their acid addition salts with pharmaceutically acceptable acids.



   The invention also provides a method of preparing an antibiotic complex of tenebrimycins, tenebrimycin I; I ', II, III, IV, V, VI or mixtures thereof, according to which a strain of Streptomyces tenebrarius is cultured in a culture medium containing assimilated carbon and nitrogen strains and inorganic salts thereof. aerobic medium in submersion 'until formation of a substantial amount of the antibiotic sought by the microorganism in this culture medium.



   The novel antibiotic complex targeted by the present invention relates to at least seven factors each having antibiotic activity. The complex in the form of free base, is soluble in water and dimethylsulfoxide slowly soluble in methanol and insoluble in most other organic solvents such as acetone, alcohols

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 higher, dioxane, ethyl acetate, diethyl ether, acet trile, methyl isobutyl ketone and hydrocarbon solvents. The free complex is stable at refrigerated temperatures, room temperature and 37 ° C for at least eight weeks over a pH range of 1 to 11.



   The infrared absorption spectrum of the antibiotic complex, obtained from a slurry in mineral oil, is shown in Figure 1 of the accompanying drawing, The bands discernible in the infrared absorption spectrum in there range from 2 to 15 microns are as follows:
3.02 (wide), 3.15, 5.82, 6.26 / u, 7.43, 8.8 / u (wide), and 9.7 / U (very wide).



   In a series of chemical tests carried out with the complex, positive reactions were obtained in the tests for ninhydrin, anthrone and the Elson test.
Morgan. The Lowry protein test gives a slightly positive reaction. The biuret and Sakaguohi tests are negative,
As indicated above, the antibiotic complex comprises at least seven distinct factors.



   In the nomenclature employed in the present description, the term mealworm I will be used to denote the antibiotic complex, while the various factors constituting the complex will be referred to as mealworm I, mealworm I, tenebrimycin II. Tenebrimycin III, Tenebrimycin IV, Tenebrimycin V, and Tenebrinyaine VI.

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  In the complex as commonly abused, tenebrimycin I, tenbrimycin I 'and tenbrimycin III are present in low amounts. Of the remaining tenbrimycins, the most abundant appears to be tenebrimycin II, which cons - titutes about 45 to 50% of the complex, and tenebrimycin V, which constitutes about 30% of the complex. Tenebrimony IV is present in amounts of about 15 to 20% while tnbrimycin VI constitutes about 10% of the complex. The most abundant tenebrimycins have been separated and characterized, the descriptions of their properties being contained in the following paragraphs.



   Mealworm II is a white solid substance. The electrometric titration in water indicates the presence of titratable groups having pK'a values of about 5.7; 6.7; 7.7; and 8.7. The specific rotation of sodium yellow light at a temperature of 25 ° C for this factor is [[alpha]] D + 159 when the concentration of the antibiotic is 1% w / v in aqueous solution. Microanalysis indicates that mealworm II has substantially the following percent composition C: 44.90; H: 8.20; N, 12.53; 0: 33.24 (direct assay). Apparent molecular weight, as provided by titration, is about 454. The empirical formula best agreeing with these data is C16H36N4O9.



   The infrared absorption spectrum of mealworm II, slurry in mineral oil, is shown in Figure 2 of the accompanying drawing. The bands discernible in the range of 2 to 15 microns of the absorption spectrum

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 infrared are as follows: 3.02; 3.14; 6.05 / u: 6.23; 7.4; 8.5; 8.75, 9.17, 9.65, 10.11, 15, 11.7 and 12.6 / u.



   Tenebrimony II forms an acetylated derivative which has a specific rotation [[alpha]] D25 = about +130, when the concentration of the compound is 1% w / v in aqueous solution. The derivative has no titratable groups remaining. Elemental composition determined by microanalysis indicates that four acetyl groups have been added by substitution into the structure of mealworm II.



   Tenebrimycin IV is a basic substance having titratable groups with pK'a values of about 5.3; 6.8; and 9.0, determined by electrometric titration in water. The specific rotation of tenebrimycin IV, determined on a 1% aqueous solution at a temperature of 25 ° C., is +114. Microanalysis indicates the following percent composition for mealworm IV: C: 41.66; H: 7.78; N: 15.02; 0: 34.93 (direct assay). The apparent molecular weight determined from titration is approximately 478. These data suggest for mealworm IV the empirical formula C16H36N5010.



   The infrared absorption spectrum of tenebrimycin IV, slurry in mineral oil, is shown in Figure 3 of the accompanying drawing. The bands discernible in the infrared absorption spectrum in the range of 2 to 15 microns are the following ; 3.05,, 17u, 5.85; 6.27 / u; 7.46 / u, 8.75 / u; 9.7; 10.5, 11.1 and 12.85 / u.

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   The tetra-aoetyl derivative of mealworm IV is a crystalline solid melting at about 265-267 C.



  The specific rotation is [[alpha]] D25 = +109 when the oonoen. tration of the derivative is 1% in aqueous solution,
The electrometric titration of tenebrimycin V indicates titratable groups with pK'a values @ about 5.5; 7.0; 8.0; and 9.1. The specific rotation of the yellow light of sodium in 1% aqueous solution for this factor, determined at a temperature of 25 ° C., is [[alpha]] D @ = + 118. The elemental composition determined by microanalysis is as follows; C: 41.05; H: 7.60; N: 23.62; 0: 36.76 (direct assay).

   The analytical data and the apparent molecular weight of 424, determined from the titration data, suggest for tenbrimycin V an empirical formula C14H32N4O10,
Figure 4 of the accompanying drawing shows the infrared absorption spectrum of mealworm V slurry in mineral oil, this spectrum exhibiting in the range 2 to 15 microns the following discernible bands 3.05 / u: 3.19 ; 6.32; 7.45; 8.75;

     9.7 / u; 11.15 / u ot 12.25,
Like mealworm IV, mealworm V forms a crystalline tetra-aoetyl derivative. The derivative melts upon decomposition at about 260 C and gives a specific rotation [[alpha]] D @ = about + 109, when the concentration of compound is 0.42% in water.



   Tenebrimony VI has titratable groups with pK'a values of about 5.6; 7.2; 8.2; and

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9.3 determined by electrometric titration in water. The assay data suggests an apparent molecular weight of
396. The specific rotation of sodium yellow light by a 1% aqueous solution of mealworm VI at a temperature
25 of 25 C is [[alpha]] D = approximately +127. The mioroanalysis indicates the following approximate percentage composition C: 44.04, H; 8.40;
N, 13.78; 0: 33.62 (direct assay). The empirical formula most consistent with the above data is C16H36N4O9.



   The infrared absorption speotre of mealworm VI is shown in Figure 5: of the accompanying drawing. The bands discernible in the range 2 to 15 microns are as follows: 3.05; 3.19; 6.35; 7.47 / u; 8.75 / u;
9.8; 12.3 and 12.95.



   A 1% aqueous solution of the tetraacetyl derivative of tenebrimycin VI has a specific rotation of about +95, at 25 C.



   Acid addition salts of mealworm or its individual antibiotic constituents can be prepared by conventional methods. Organic or inorganic acids can be used for salt formation. A suitable method for the preparation of These salts consist in adding a solution of the salt-forming acid to an aqueous solution of the antibiotic. In the case of insoluble aid addition salts, the salt precipitates from the solution and is easily separated by conventional techniques such as filtration or centrifugation. In the event that the acid addition salt

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 EMI8.1
 



  1 TABLEAUt CI, -ror-iat (izra.-14e Alt- IELR of tenebrirny es and known antibiotilues a, 3rant
 EMI8.2
 similar properties
 EMI8.3
 % Solvent system and ne value -
 EMI8.4
 
<tb> A <SEP> B <SEP> C <SEP> D <SEP> E <SEP> F <SEP> G
<tb>
 
 EMI8.5
 Tenebrilcycin coaplex - 0.65 0.32 0 0 Tenebri :: 3ycin I 0.20 Tenebrimycin 1 '0.27 Tenebrimycin II 0.40 o, 17 0.32 Tenebrimycin III 0.40' 0 ,,} 4 0.41 - Tenebrimycin IV o, 49 0120 0.32 TénibrimYc1ne V 0.55 0122 0.35! "N'brimyc1ne VI 0.71 0131 0.38 - xana.myc1ne 0.35 0.35 o.41 0.65 0.42 0 0 X.c1n. B 0.57 o, 2É '0.32 0170 0329 0 0.65 0.51 0.39 04tntamyo1ne 0.83 0.75 0.62 - 0151 0 0 ,.



  0.95 c-Unu11ne o, 44 0.25 0.34 0.65 0.35 0 0, Jl4omyo1De 0.60 0.12 oig 0 0.05

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 EMI9.1
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The mealworm complex and the individuela factors which constitute the said complex have an inhibitory action against the growth of microorganisms which are pathogenic to animal and plant life. Inhibitory concentrations of the complex and individual tenebrimycins against a number of organisms are indicated in the following table The minimum Inhibitory concentrations are determined by the agar dilution method.

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 EMI11.1
 



  1 A BLE A -.! L.JI
 EMI11.2
 03 R-A .RjH-naj. minimum concentration / ìbitrix ¯¯Qzuy ± 1 Tnebrimycinea Tenebrimycin II Tenebrimycin IV Tenebrinycin V Tenebrimycin VI Stapbylococeus aureus 3055 6.25 6.25 6.25 6.25 3.12 Ba fus subtilis 1.56 .c 1.56 <1.56 <1.56 G '.56 M3ib.eterium avium - <' L, 56 <1.56 = -1.56 6.25 1.56 Sr2ococcus faecalis 6.25 3.12 1.56 3112>, 12 Lactobacillus case ! 25 50 25 ^ 25 32;

  5
 EMI11.3
 
<tb> Leuconostoo <SEP> citrovorum <SEP> 25 <SEP> 50 <SEP> 25 <SEP> 25 <SEP> 12.5
<tb> Escherichia <SEP> coli <SEP> No. <SEP> 1 <SEP> 25 <SEP> 25 <SEP> 12.5 <SEP> 12.5 <SEP> 12.5
<tb> Escherichia <SEP> coli <SEP> No. <SEP> 2 <SEP> 25 <SEP> 50 <SEP> 12.5 <SEP> 25 <SEP> 12.5
<tb> Proteus <SEP> sp. <SEP> No. <SEP> 1 <SEP> 50 <SEP> 100 <SEP> 50 <SEP> 50 <SEP> 25
<tb> Proteus <SEP> sp. <SEP> No. <SEP> 2 <SEP> 25 <SEP> 25 <SEP> 12.5 <SEP> 25 <SEP> 12.5
<tb> Pseudomonas <SEP> sp. <SEP> No. <SEP> 2 <SEP> 25 <SEP> 25 <SEP> 100 <SEP> 100 <SEP> 6.25
<tb>
 
 EMI11.4
 Pseudomonas sp. Nc. 5 6.25 12.5 6.25 3.12 C -1.56
 EMI11.5
 
<tb> Klebsiella-Aerobacter <SEP> No. <SEP> 14 <SEP> 6.25 <SEP> 12.5 <SEP> 3.12 <SEP> 12.5 <SEP> 6.25
<tb> Klebsiella-Aerobacter <SEP> no, <SEP> 15 <SEP> 12.5 <SEP> 12.5 <SEP> 6.25 <SEP> 12.5 <SEP> 6.25
<tb>
 
 EMI11.6
 Salmonella sp.

   No. 1 '25 25 25 25 12.5

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   TABLE II (continued)
 EMI12.1
 yibrio metschnikovii 12.5 12.5 12.5 6.25 6.25 -Agrobacterium tumefaciens 50 25 25 50 50 Erwinia amylovora <1.56 4 1.56 - 1.56. 1.56 <1.56 at Pseudomonas solanacearum 25 25 12.5 12.5 .2.5 .DLanthomonas phaseoli z 12.5 12.5 6.25 3.12 The minimum inhibitory concentrations for individual tenebrimycins are determined with the bases free. The tenebrimycin complex is used as a sulfate.

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   The acute toxicity of mealworm complex and its relatively more abundant constituents was determined in mice. The LD50 values of the sulphate, tenebrimycin are about 300 mg / kg when the antibiotic is injected intravenously and about 350 mg / kg when the drug is administered intraperitoneally.

   At subcutaneous doses of 750 mg / kg or oral doses of 5600 mg / kg, all mice survived. No noticeable irritation was observed when four rabbits were each treated with a drop of 50% aqueous solution of mealworm in one eye three times daily for five days. LD50 values, expressed in mg / kg of the free base, for the individual tenebrimycins administered intravenously to mice are as follows; Tenebrimony II, about 385; Tenebrimycins IV, approximately 220; tenebrimycin V ,. about 140;

   and tenebrimycin VI, approximately 120,
The novel antibiotic complex according to the present invention is produced by culturing an appropriate strain of an organism of the actinemycer class under aerobic conditions in a suitable culture medium until the culture medium contains an antibiotic activity. notable. Tenebrimony can be recovered using various isolation and purification techniques known to those skilled in the art.

   The antibiotic complex can be used as is or be subjected to other purification processes to isolate its various constituents in a higher state of purity,

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Due to the uncertainty of taxonomic studies with organisms of the Streptomyces group, it. There is always an element of doubt associated with the classification of a newly discovered organism. However, as far as can be determined, the organism which produces the antibiotic complex of the invention does not sufficiently resemble any of the Streptomyoes species described by Waksman in The Aotinomyoetes, Vol. II, The Williams and Wilkens Company (1961), to ensure a comparison of cultures. The organism is therefore described and characterized as a new species.



   The organism producing the antibiotic complex of the invention is a spiral-forming, heat-resistant, aerobic to mioroaerophilic streptomyoes with oblong smooth-walled spores. It is the only one to be inhibited by relatively weak intensities of artificial light.Because of this last property, we have chosen for this organism the new species name Stres-
 EMI14.1
 tommees tenebrar1us SPI n.



   The organism was isolated from a soil sample by suspending the soil in sterile distilled water and spreading the suspensions on nutrient agar. The seeded nutrient agar plates are incubated at 25-35. C until growth is assured. At the end of the inaubation period, colonies of the antibiotic-producing organisms are transferred to agar slants by means of a platinum loop.

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  The inolinated agars are then incubated to provide suitable amounts of inooulum for the production of the antibiotic. The strain of the organism used for the production of the antibiotic complex is placed in permanent deposit in the Amerioan Type. Sulfide Collection in Washington, and has been assigned the ATCC Culture # 17920.

   Said strain produces all the factors of the antibiotic complex, but under preferred fermentation conditions, tenebrimycins II, IV, V and VI are predominant,
The methods employed in taxonomic studies of S. tenebrarius ATCC 17920 are commonly used in the taxonomy of actinomycetes. The culture characteristics are observed after 14 days of incubation. The morphology is determined on Czapek peptone agar and Bennett agar during an incubation of 2 to 7 days.

   We observe the action on milk and the reduction of nitrate at 7 and 14 days, the production of hydrogen sulfide at 24 and 48 hours, and the use of carbon at 10 days.



  Unless otherwise indicated, cultures are incubated at 37 C.



   The carbon utilization tests are carried out according to the method described by Pridham and Gottlieb, in J. Bact., 56, 107 (1948).



   The results of the taxonomic studies are summarized in the following paragraphs. The numbers in parentheses refer to the color blocks defined by Maerz and Paul, in Dictionary of ColQr, McOrax Book Company (1950). The colors represented by the color blocks

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 have been translated to ISCC-NBS color names found in Circular No. 553. U.S. Department of Commerce, National Bureau of Standards, 1955.
 EMI16.1
 



  Morphology mJypsop.ueJyoaactr1stiQues eulture et h siolo ie of S tenebrarius ATCC 1 920
Morphology'- On a peptone-Czapek agar medium, formations of branched spores are formed in tufts on the aerial mycelium. Isolated spores are rare.



  The intact aerial myoelium is easily detached from the substrate. The chains of mars spores usually form 5-6 open spirals. The spores are oblong to cylindrical. When viewed through the electron microscope, the spores appear smooth and measure 0.7 to 1.3 microns by 2.0 to 2.1 microns. Hyphae clusters are observed on Bennett's medium,
 EMI16.2
 Characteristics of the colonies m111 $ u êJose - Czapek - Sparse growth; sparse aerial mycelium, pale yellow-orange (11-A2); good sporulation; pale yellow-orange reverse (11-A2); slightly pink soluble pigment (1-B1).



   Peptone-Czapek medium - abundant oroissanoe; aerial mycelium abundant, light yellowish brown with white areas (11-A4); sporulation abundant; light greyish-red reverse (4-Hl); grayish-pink soluble pigment (4-B1).



   Calcium Malate Agar - moderate growth, moderate aerial mycelium, pale yellow-orange (11-A2); sporulation

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 moderate, pinkish-gray reverse (4-D1); soluble pigment pinkish-pink (4-Bl).
 EMI17.1
 Tyrosine agar - sparse growth; sparse aerial mycelium, pale yellow-orange (9-B2); scant sporulation; pale yellow-orange reverse (10-B3); no soluble segment.



   Mineral Salts - Starch Agar - moderate growth, moderate aerial mycelium, pinkish-brownish with white areas (11-A4); profuse sporulation; reverse pale yellow (11-B2); soluble pale yellow pigment (11-B2).
 EMI17.2
 



  Moderate glucose-aspara1ne-oroissanoe-glucose agar, moderate aerial mycelium, pale yellow (9-D2) with white areas (10-A1); moderate sporulation; pale yellow (11-B2); no soluble pigment.



   Tomato-oatmeal paste - abundant growth, abundant aerial mycelium, light yellowish-brown (12-B5); profuse sporulation; reverse dark greyish brown-red (48J2); dark purple red soluble pigment (47-H1).



   Yeast extract - abundant growth, abundant aerial mycelium, pale yellow-orange (11-A2) with white areas; profuse sporulation; moderate yellow reverse (11-J6); no soluble pigment,
 EMI17.3
 g4] p.a, Q, flg ± aojàw ± ,,, - sparse growth, sparse aerial yod- lium white (10-Al); scant sporulation; reverse greyish-yellow (12-12); no soluble pigment.

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 EMI18.1
 MiliéiM-Bennett - moderate growth, moderate air myaeliut blano (10-Al to 10-Bl); moderate sporulation; reverse pale yellow (11-C2); no soluble pigment,
Action on milk - fonoe yellow growth ring on the surface. Coagulation and peptonization observed.



   Nitrate reduction - Positive
H2S production - negative,
Nutritional gelatin - Total liquefaction after 14 days,
Temperatures required on PeptoneCzapek agar medium -
20 - no growth,
26 - good growth, no aerial mycelium.



   - Moderate but sporulating aerial growth and mycelium.



   37 - oroissanoe, aerial mycelium and sporulation, abundant.
 EMI18.2
 



  43 * - abundant growth # mycelium and sporulation.



   50 - abundant growth, aerial mycelium and sporulation.



   55 - sparse growth.



   60 - no growth.



   Lethal temperature. Spores in a spore suspension heated to 75 C for 15 minutes remain viable. When the spore suspension is heated to 100 C for 15 minutes, no viable spores remain,

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Oxygen Tension - Growth is aerobic or microaerophilic in subcultured cultures.



   Effect of ferric ion - Red soluble pigment occurs, only in the presence of ferric ion, and the intensity of pigmentation is proportional to the concentration of ferric ion in a certain range.
 EMI19.1
 



  Observed effect-of-ph on Zelose-peptone-Czapek medium
No growth below pH 5.0; pH 5.0-6.0 good growth and good aerial mycelium. Growth and sporulation abundant at pH 7.0; pH 8.0 to 8.6, good growth and good aerial mycelium. The soluble pigment is most intense at pH 5.0-6.0 and slight to zero between pH 6.5 and pH 8.6.



   Light reaction, observed on Czapek agar medium
Growth and sporulation are abundant in obs- ourity, When plate ulcers are incubated 38.1 cm from a standard 15 watt cold-white fluorescent light source, growth is scant and aerial mycelium is absent . Growth and aerial mycelium are moderate when the plate cultures are Incubated 38.1 cm from a 60 watt tungsten filament frosted bulb,
The results of the carbon utilization test performed with S. tenebrarius ATCC 17920 are summarized in the following Table III. In this table, the symbols are interpreted as + = positive.

   (+) = probable (-) - doubtful - - null

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 TABLE III
 EMI20.1
 Use of carbon by S tenebrarius strain ATCC N 7. 20
 EMI20.2
 ..., ... ",:," 't',; w.



  Srou: :( "&. E 9a: r.! O n 80ucce jēcarbonj, Answer
 EMI20.3
 
<tb>
<tb> L (+) <SEP> Arabinose <SEP> - <SEP> D (+) <SEP> Trehalose <SEP> +
<tb> L <SEP> (+) <SEP> Rhamnose <SEP> - <SEP> L (+) <SEP> Raffinose <SEP> D (-) <SEP> Ribose <SEP> + <SEP> Cellulose
<tb> D <SEP> (+) <SEP> Xylose- <SEP> Inulin <SEP> (-)
<tb> D (-) <SEP> Fructose <SEP> + <SEP> i-Inositol <SEP> +
<tb> D (+) <SEP> Mannose <SEP> + <SEP> MannitolD (+) <SEP> Glucose <SEP> + <SEP> d-Sorbitol <SEP> (-)
<tb> Lactose <SEP> (-) <SEP> Salicin <SEP> '<SEP> (+)
<tb> Sucrose
<tb> Maltose <SEP> + <SEP> control <SEP> (without <SEP> carbon) <SEP> (-)
<tb>
 
S, tenebrarius ATCC 17920 produces, in addition to mealworm, a known antibiotic and antifungal, oaerulomyoine which is also produced by Streptomyces
 EMI20.4
 caerullus -Cano J. Microb101.,, 31? (959) ", /.



   A study of a culture of the latter organism revealed that it does not produce mealworm under the conditions described for its culture, and that it cannot be induced to produce it under various conditions.



   A second source of the new species
 EMI20.5
 Streptomyces tenebrarius SJ2, n - ,,, a source which produces only three of the factors of the antibiotic complex, has also been isolated and characterized. It has been attributed to this source of S, te-
 EMI20.6
 nebrarius which produces only mealworms 1, '1' and

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II, ATCC order number 17921. Source ATCC 17921 says, prides itself on source ATCC 17920 mainly in that it does not produce aerial mycelium or spores, and it does not produce soluble pigment on the body. most backgrounds,
 EMI21.1
 do5aaée de ca. ,, 'actt :(' i8atiorl for this source of the body shooting question xonl summarized in the following paragraphs.

   The methQu-., Used in the taxln.cm1ques studies of S. tenebrarLLB ATCC 17921 are the rings as those referred to above for the description of the characterization of S, ten-
 EMI21.2
 brar3.us ATCC 1,324, and the symbols used have the same meaning as above.



  Microscopic methodology. Cuture and phs1010gia characteristics of S. tenebrarlus ATCC 1J9Ql¯ Morphology. There are neither aerial mycelium nor spores; vegetative mycelium Fragments in small quantities, Characteristics of colonies.
 EMI21.3
 Medium-Zelose-Cza2ek- Good vegetative growth, yellowish-white (9-Bl); no aerial myoelium, no soluble pigment,
 EMI21.4
 Medium, peptone-Czapek - Abundant vegetative growth, greyish yellow (12-B3); no aerial mycelium, slight brownish soluble pigment.
 EMI21.5
 Ce malate agar, u - Moderate vegetative growth, greyish yellow (12-B2); no aerial mycelium;

   light grayish-brown soluble pigment,,

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Tyrosine agar moderate vegetative growth, greyish yellow (11-B2); neither aerial myoelium nor soluble pigment.



   Agar with mineral salts-starch-. Good vegetative mycelium, light yellow-brown (13-c6); no aerial mycelium; light brown soluble pigment.



   Agar-Glucose-Asparagine - Moderate vegetative growth, greyish yellow (11-D2); no aerial mycelium or soluble pigment.



   Tomato-oatmeal paste - Moderate vegetative mycelium, pale yellow (10-B2); no aerial myoelium or soluble pigment.



   Yeast extract - Moderate vegetative mycelium, light yellow-brown (12-E5); no aerial mycelium or soluble pigment,
Nutrient Agar - Moderate vegetative growth, light yellow (9-K4); no aerial myoelium or soluble pigment,
Benhett Agar Medium - Moderate greyish yellow (12-E4) vegetative growth no aerial mycelium or soluble pigment.



   Action on milk - Buff colored growth ring on the surface. Coagulation and peptonization.



   Nitrate reduction - Positive
H2S production - Negative
Nutritional gelatin - Total liquefaction in 7 days,
Oxygen Tension - Growth is aerobic or microaerophilic in subcultured cultures.

 <Desc / Clms Page number 23>

      
 EMI23.1
 



  Temperatures Required on Bennett's Medium - Growth is moderate at 26, 30, 43 # 49 and 5500 and most abundant at 37 C; no growth at 60 C. Vegetative mycelium is pale yellow-brown at 37 C and below and slightly reddish-brown at 43 C and above. Neither aerial mycelium nor soluble pigment at any temperature,
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Although the present invention is described in detail with particular reference to the recently discovered S.

   tenebrarius ATCC 17920 and ATCC 17921, it is understood that the production of the tenebrimony complex or of the various faoteurs oonstituent said complex by other strains or mutants of said organisms, come within the scope of the invention.

   The proportions of the various factors tenebrimycin
 EMI23.7
 pMducM by other strains or mutants are not required.

 <Desc / Clms Page number 24>

 sarly the same as those indicated in the present description. Other strains or mutants can be produced or obtained by known methods, for example by subjecting an organism producing tenebrimycin to irradiation by X-rays or ultraviolet or to chemical agents such as for example isothiocyanates:
The culture medium which can be used for the production of mealworm can be selected from several media since, as it appears from the use tests described above, the mealworm-producing organisms are capable of using a variety of media. sources of energy.

   However, for economy of production, maximum yield of antibiotics and ease of isolation, certain culture media containing relatively simple nutrient sources are preferred. For example, media which are useful for the production of mealworm include an assimilable source of carbon such as glucose, fruotose, mannose, maltose, starch and the like. In addition, the media which can be used contain an assimilable nitrogen source such as peptones, hydrolyzed casein, yeast, amino acids and the like. The presently preferred sources of nitrogen are peptones, hydrolyzed oasein and glutamine.



   Inorganic salts, for example those providing oleum, magnesium, sodium, potassium, chloride, sulfate, phosphate and carbonate ions, can be incorporated into culture media with favorable results, although an excess of phosphate should be avoided. because it seems to decrease the yields

 <Desc / Clms Page number 25>

 antibiotic. Growth factors such as yeast or yeast extract can also be advantageously incorporated into the medium.



   As is necessary for the growth and development of other microorganisms, the growth medium used to grow the microorganisms used in the present invention must also contain the essential trace elements. Trace elements are commonly present as accidental impurities at the time of addition of other constituents of the medium.



   The conditions of submerged aerobic culture are the conditions of choice for the production of mealworm. For the preparation of relatively small amounts, shake flasks and surface culture can be used, but for the preparation of large amounts, aerobic culture submerged in sterile reservoirs is preferred. The medium in the sterile reservoir can be inoculated with a sporulated suspension, but due to the growth retardation that occurs when using a sporulated suspension as the inoculation, the vegetative form of the culture is preferred. By thus avoiding growth retardation, efficient use of the fermentation apparatus is achieved.

   Consequently; it is desirable to first produce a vegetative inoculum of the organisms by inoculating a relatively small amount of culture medium with the spore form of the organism when a vegetative inoculum has been obtained

 <Desc / Clms Page number 26>

 young active person, to transfer the vegetative inoculum under aseptic conditions into the large reservoir. Advantageously, an aliquot of the vegetative inooulum is used, equal to approximately 4% of the volume of the medium in which the inooulum is induced. The fermentation medium in which the vegetative inoculum is produced may be the same or different from the medium used for the production of tenebrimycin on a large scale.



   As is evident from the details of the temperatures required for the organism given above, the organism will grow in a relatively wide range of temperatures. However, it appears that organisms grow best at temperatures in the d range. 'about 30 to 50 C.



  It appears that the optimum production of tenebrimycin is at a temperature of about 37-43 C. The organisms that produce mealworm are sensitive to light and do not grow well in its presence. Consequently, the fermentations employing the organisms are advantageously carried out in the absence of visible light.



    'In accordance with the usual practice in aerobic submerged culture processes, sterile air is introduced into the packing medium. For efficient removal of the organisms and good production of tenebrimycin, the volume of air employed in the production. in a mealworm tank is more than 0.1 volume of air per minute and per volume of culture medium, and preferably significantly higher.

   Efficient growth of organisms and yields

 <Desc / Clms Page number 27>

 optima of mealworm are obtained when the volume of air used is at least one volume of air per minute and per volume of culture medium,
The concentration of mealworm activity in the fermentation medium can be easily monitored during fermentation by assaying the inhibitory activity of samples of the culture medium against the growth of organisms. known to be inhibited in the presence of tenebrimycin. Two of the organisms thus employed to monitor tenebrimycin production are Xlebsiella pneumoniae and Mycobacterium butyricum.

   The former organism is generally used in the well-known turbidimetric technique, while the latter is used in the bucket plate method,
In general, the maximum production of the antibiotic
1 occurs after about 4-7 days post / inoculation of the culture medium when using the submerged aerobic culture, or the shake flask culture and a little longer when using the surface culture.



   Mycelium and non-diaaoua residues are separated from the fermentation broth by standard methods such as filtration or centrifugation. Antibiotic activity is contained in the filtered broth and can be recovered using adsorption techniques. The adsorbents which can be used most advantageously are the exchange resins. cationic agents, for example those available commercially under the name "IRC-50".

 <Desc / Clms Page number 28>

 



   In general, the process for recovering mealworm from fermentation broth is as follows. All of the broth is filtered using a filter aid, after having lowered the pH of the medium to approximately pH 2 by adding an acid, such as sulfuric acid, phosphoric acid, hydrochloric acid and the like. The pH of the filtrate is adjusted to about pH 5.5 by addition of a concentrated base and the mixture is filtered again. The filtrate is passed through an ion exchange column packed with a resin such as IRC-50 in the ammonium form. The column is then washed with distilled water and the antibiotic activity is eluted with dilute acid. The fractions containing the antibiotic activity are combined and concentrated to about 1/20 of the original volume.



   The pH of the concentrate is raised to about pH 11 by the addition of a concentrated base and the basic concentrate is poured into about 6 volumes of acetone, mixed well and cooled. The microbiologically inaotive precipitate which separates is removed by filtration. The pH of the filtrate is lowered to approximately pH 3.5 by the addition of 20% sulfuric acid with vigorous stirring,
The tenebrimycin complex is precipitated in the form of the sulfate, while the caerulomycin obtained as a by-product in the fermentation flows into the supernatant layer, which is discarded.



   Tenebrimony sulfate is dissolved in a minimum amount of water and the aqueous solution is passed through a Dowex 1x1 column. The aqueous solution of tenebrimycin sulfate is followed on the column by distilled water and

 <Desc / Clms Page number 29>

 collects the column tributary in various fractions. The active fractions are combined and concentrated into a thick syrup and then dried to give the free base of mealworm.



   The main factors constituting the tenebrimycin complex can be obtained if desired in the form of simple antibiotics by subsequent fractionation of the mealworm obtained above on ion exchange columns. For processing, mealworm is used in the form of an acid addition salt, preferably sulfate. A solution of tenebrimycin sulfate is suitably obtained by preparing a 20% aqueous solution of tenebrimycin and adjusting the pH of the solution to about pH 4.5 by adding sulfuric acid. Colored impurities can be removed by stirring the solution thus prepared with about 5% (weight / volume) of an activated charcoal such as for example "Darco G-60".

   Effective decoloration is usually obtained by stirring the mixture containing the coal for about an hour. The mixture is then filtered and the filtrate which contains the mealworm sulfate is loaded onto a column packed with IRC-50 resin in ammonium form. The column is washed with water and the individual antibiotics constituting the tenebrimycin complex eluted with 0.1N ammonia. In general, the elution is carried out extremely slowly and it usually takes a very large number of fraotions to effect the fractionation of the complex. Thus, for example, the total fractionation.

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 of about 250 g of mealworm requires about 700 fractions, each of about 900 ml of eluate.



  . The first active fractions obtained contain tenebrimycins I, I 'and II. These are followed by a series of active fractions in which tenebrimony II is the only antibiotic present. A number of inactive fractions followed elution of mealworm II before fractions containing mealworm IV were eluded. It appears that the first fractions containing mealworm IV contain traces of tenebrimycin III.



  Tenebrimycin V is obtained off the column in the active fractions which follow mealworm IV. To obtain Tenebrimycin V, the column is subsequently eluted with a more concentrated ammonia solution after all of the Tenebrimycin V has been collected from the column. A suitable eluent for the separation of mealworm VI is 0.3N ammonia.



   The following examples illustrate the present invention without, however, limiting its scope: Example 1.



   Preparation of mealworm
A sporulated culture of S. tenebrarius ATCC 17920 is produced by growing the organism on a modified Bennet's inolin agar, having the following composition:

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 Dextrin 10 g Yeast extract (Difoo) 1 g Hydrolyzed casein ("NZ-Amine A", from Sheffield Farms) 2 g Beef extract (Difoo) 1 g
 EMI31.1
 CoCl, 6t0 001 g
Agar, washed 20 g
Deionized water, q, s, p, 1000 ml.



   The medium is adjusted to pH 7 before switching to au * t oolave,
The medium is inolated with spores of S. tenebrarius ATCC 17920 and incubated in the absence of visible light in @ five days at 37 ° C. The growing culture is covered with water and the agar gently scraped to remove the spores and. provide an aqueous suspension of spores.



   The suspension of spores thus obtained is used to inolate 800 ml of a medium having the following composition
Dextrose 0.05%
Flour "Nutrisoy" (Archer -Daniels-Midland Company containing 35-45% dispersible protein 1.5% "Dextrin 700" (low chloride potato dextrin from Morningstar-Paisley
Company 1%

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 potassium chloride 0.1% "NZ-Amine A" 0.3% KH2PO4 0.05% MgSO4.7H2O 0.5% CaCl2, 2H2O 0.025 Deionized water q.s.p.



   The inoculated vegetative medium is incubated at approximately 37 ° C. for 16 hours on a rotary seooueuse operating at 250 revolutions per minute and having a stroke of 6.35 µm.



   A 50 ml potion of the vegetative culture is used to inoculate a 44 liter seeding tank containing an aqueous medium having the following composition: Dextrose 1% Soybean 1.5% KH2PO4 0.05% MgSO4 0 , 5%
KCl 0.1%
CaCl2,2H2O 0,025% antifoaming agent 0,025%
The medium of the seeding tank is sterilized at 120 ° C. for about 30 minutes, The inoulated medium being inoubted at 3700 for 12 hours, Agitation is started at 370 revolutions per minute immediately after inoculation and the inoculation is maintained. aeration at a rate of 22.65 liters per minute during the incubation period.

   At the end of the incubation period, the contents of the seed tank are used.

 <Desc / Clms Page number 33>

 to inoculate a 945 liter fermentation tank containing
 EMI33.1
 a medium having the oomoe1t1on touted Dextrose 4% refined soybean oil 3% soybean flour 3% NH4Cl 0.5% CaCl2 0.3% MgSO 0.2% NH4NO3 0.1% "NZ (Amine A" 0.5 % antifoam agent 0.2
 EMI33.2
 Water without q.s.P.



   Before inoculation, the fermentation medium is sterilized for 30 minutes at 120 C. Fermentation is
 EMI33.3
 carried out at 37 * 0 with aeration at a flow rate of 522.16 liters / minute in the period between inoculation and re-
 EMI33.4
 oaltej We start stirring at 125 revolutions / minute and increase it. Lies up to 180 rpm after 12 hours. We continue the fermentation for five days
The fermented sulfide broth is filtered to separate the mycelium and other undissolved solids.

   The
 EMI33.5
 broth filtered to a maximum concentration of about 680 units per milliliter. Example 2
 EMI33.6
 h "d, bAnààÀ aa4 # AlSà-àni #" xi "> UlLàà à bTàX 'The process described in Example 1 is followed;

 <Desc / Clms Page number 34>

 using S. tenebrarius ATCC 17921 as an antibiotic-producing organism. The filtered fermentation broth thus obtained contains mainly tenebrimycin II with lower proportions of tenebrimyoins 1 and I '.



    Example 3
Isolation of the mealworm complex
The pH of 880 liters of antibiotic fermentation broth obtained as described in Example 1 is lowered to about pH 2 by adding about 10 to 15 liters of 20% aqueous sulfuric acid. After adding 45 kg of a. Aoidified broth commercial filter aid, mixed vigorously / filtered. The filter cake is washed with water and the washing solution is added to the filtrate.



   The pH of the filtrate is adjusted to 5.5 by adding 50% aqueous sodium hydroxide. About 4 kg of oommeroe filter aid is added and the mixture is stirred vigorously and filtered; The filtrate was passed through a 10.16 µm x 1.83m IRC-50 resin column. The column is packed with resin in ammonium form to a height of 111 to 114 cm. When all the filtrate has passed through the column, the column is washed with about 200 liters of distilled water. The antibiotic activity is then eluted in a period of about 12 to 15 hours with about 150 to 175 liters of 0.1 N sulfuric acid, the eluate being collected in fractions of 10 liters. Antibiotic activity becomes detectable in the eluate when its pH drops to about pH 4 or slightly lower.



  As the elution progresses the pH of the eluate continues to drop

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 until it reaches a value of approximately pH 1.5. The fractions containing the antibiotic activity are combined and concentrated under reduced pressure to about 1/20 of the initial volume.
The pH of the concentrated eluate is adjusted to pH 11 by addition of 50% aqueous sodium hydroxide and the basic oonoentrate is then added to approximately 6 volumes of acetone. The mixture is cooled and allowed to stand to separate a precipitate free from antibiotic activity. The mixture is filtered and the filter cake containing the inactive precipitate is washed with water and discarded.



   The pH of the filtrate is adjusted to pH 3.5 by the addition with vigorous mixing of 20% aqueous sulfuric acid.



  The mealworm complex precipitates as sulfate during this treatment and is substantially completely precipitated when the pH reaches about pH 4 or slightly lower. The mixture is filtered and the filtrate containing oerulomyoine co-produced in the fermentation is discarded.



   The filter cake consisting of tenebrimony sulfate is dissolved in the minimum amount of water and cooled; Qh discards solid substances which do not dissolve easily, as well as solids which precipitate or crystallize during cooling. The clear solution of tenebrimycin sultate is passed through a resin bed of 1 x 16 cm x 2.17 m owex resin 1 x 1 in basic form,

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The antibiotic solution is followed on the column by about 40 to 50 liters of distilled water,

   The effluent is collected in a 10 liter fraction and the active fractions are combined and concentrated in vacuo into a thick syrup which is dried in a flat basin to give the dry tenebrimycin,
Example 4
Tenebrimycin Factor Separation
Tenebrimycin sulfate is obtained by adding the pH of a 20% aqueous solution containing 258 g of the complex. of mealworm in the form of free base at pH 7.5 by addition of concentrated acid ". The solution of tenebrimycin sulfate thus prepared is decolorized by stirring at about 1 hour with 5% (weight / volume) of Darco G-60 The solution is filtered and the filtrate is passed through a 9 x 1050m column packed with about 7 liters of IRC-50 resin in ammonium form.

   When the filtrate containing the antibiotic salt has passed over the column or begins to elute the antibiotic retained by the column with a 0.1 N ammonia solution at a flow rate of about 20 ml per minute. The eluate is collected in fractions of about 900 ml and the miorobiological activity is assayed in each fraction. The fractions containing the same antibiotic factor are combined and lyophilized to obtain
 EMI36.1
 seohe individual endbrimyoines.

   The individual tenebr1myo1nes are distributed among the aotive fractions as follows,

 <Desc / Clms Page number 37>

 
 EMI37.1
 Fta1 (9n nti.19Qve "ése ,, t 1-34 Inactive 35-53 Tenebrimony I, I 'and II 54-77 Tenebrimony II 78-153 Inactive 154-172 Tenebrimycin IV with a trace @ of
Tenebrimony III 173-249 Tenebrimony IV 250-256 Inaotives 257-634 Tenebrimony V 635-6681 Inaotives 669-698 Tenebrimony VI 1) at this time, the eluate is replaced by 0.3 N ammonia,

     Example
 EMI37.2
 Preparation of mealworm hellanthates
The preparation of the helianthates of individual tenebrimyoins is illustrated by the following process for the preparation of tenebrimycin II helianthate.



   . A solution of 2 g of tebrimony II in 22 ml of water is prepared and adjusted to pH 5 by the addition of 50% sulfuric acid. A hot 10% aqueous solution of methyl orange is then added to the antibiotic solution. Tenebrimony II helianthate separates out as a precipitate and is separated by filtration. The solid salt is washed vigorously with hot water and dried.

 <Desc / Clms Page number 38>

 under vacuum to give 3.5 g of mealworm II helianthate.



  Example 6
Final purification of tenebrimycin factors
The final purification of individual mealworm factors is illustrated by the following method for the purification of mealworm VI.



   Tenebri-, myoin VI helianthate is prepared according to a process analogous to that described in Example 5. 4 g of the salt thus prepared are stirred with approximately 200 ml of water adjusted to pH 2 by addition of acid. sulfuric. The mixture is stirred for about 4 hours and then filtered through a sintered glass of medium porosity to separate the helanthic acid. The filtrate is decolorized by passing through a thin layer of activated carbon and then passing it over a medium. 1 x 32 cm column packed with Dowex 1 x 2 resin in hydroxyl form. The filtrate is followed on the column with distilled water to complete the elution of the antibiotic activity.



   The effluent is collected in fractions and the active fractions are combined and lyophilized to give a dry residue consisting of the antibiotic. The residue is dissolved in about 15 ml of water and then passed through a 1.25 cm column packed with Bio Rad AG11A8 resin (ion-retardant type resin prepared by polymerization of acrylic acid. in a Dowex 1 resin, sold by Bio Rad Laboratories,
Richmond, California). The antibiotic activity is eluted from the column with distilled water and the active fractions are again combined and lyophilized to give about 700 mg of mealworm VI of high purity.

 

Claims (1)

CLAIMS. 1.- Antibiotic substance, characterized in that it comprises a complex of tenebrimycins or the antibiotics distinct from this complex or the mixtures of these distinct antibiotics or the addition salts of acids which they form with acids pharmaceutically acceptable. 2, - Antibiotic according to claim 1, characterized in that the active agent is the complex of tenebrimycins, tenebrimycin 1, tenebrimycin 1 ', tenebrimycin II, tenebrimycin 111, tenebrimycin IV, tenebrimycin V, tenebrimycin VI or a mixture of these tenebrimycins. 3.- Antibiotic according to claim 1 or 2, characterized in that the active agent is the complex of tenebrimycins which has the following properties: the complex is a white solid substance which is soluble in water and dimethylsulfoxide, slowly soluble. in methanol and insoluble in most other organic solvents; the complex is stable at pH 1 to 11, the complex gives a positive reaction to ninhydrin, anthrone and Elson-Morgan, a slightly positive reaction to Lowry protein and negative reactions to biuret and Sakaguchi and the complex show discernible absorption maxima in the infrared spectrum at 3.02, 3.15, 5.82, 6.26, 7.43, 8.8 and 9.7 microns, 4. Antibiotic according to claim 1 or 2, characterized in that the active agent is tenebrimycin 11 which has the following properties: tenebrimycin It is a white solid substance whose titratable radicals of pKa d about 5.7, 6.7, 7.7 and 8.7 measured by electrometric titration in water; tenebrimycin 11 has a molecular weight <Desc / Clms Page number 40> apparent circle of approximately 454 measured by electrometric titration; Tenebrimycin II has a specific rotation [[alpha]] D25 of + 159 (c = 1% w / v); tenebrimycin II has the following approximate composition: C 44.90%, H 8.20%, N 12, 53% and 0 33.24% and exhibits absorption maxima in the infrared spectrum (on suspension in mineral oil), at 3.02, 3.14, 6.05, 6.23, 7.4, 8.5, 8.75, 9.17, 9.65, 10.11, 11.15, 11.7 and 32.6 microns. 5.- Antibiotic according to claim 1 or 2, characterized in that the active agent is tenebrimycin IV which has the following properties: latenebrimycin IV is a white solid substance, the titratable radicals of which have pKa of approximately 5.3, 6.8, 7.8 and 9.0 measured by electrometric titration in water; Tenebrimycin IV has an apparent molecular weight of 478 as measured by electrometric titration; enebrimycin IV has a specific rotation [[alpha]] D25 of- + 1140 (c = 1% w / v); Tenebrimycin has the following approximate composition: C 41.66%, H 7.78%, N 15.02% and 0 34.93%; and exhibits absorption maxima in the infrared spectrum (on suspension in mineral oil) at 3.05, 3.17, 5.85, 6.27, 7.46, 8.75, 9.7, 10.5, 11.1 and 12.85 microns. 6.- Antibiotic according to claim 1 or 2, characterized in that the active agent is tenebrimycin V which has the following properties: tenebrimycin V is a white solid substance whose titratable radicals have pKa of about 5.5 , 7.0, 8.0 and 9.1 measured by electrometric titration in water, Tenebrimycin V has an apparent molecular weight of approximately 424 measured by electrometric titration, Tenebrimycino V has a rotating specific [[alpha]] D25 of +118 (c - 1% w / v); mealworm has the following approximate composition: <Desc / Clms Page number 41> C 41.05%, H 7.60%, N 13.62% and 0 36.76% and exhibits absorption maxima in the infrared spectrum (on suspension in mineral oil) at 3.05.3, 19, 6.32, 7.45, 8.75, 9.7, 11.15 and 12.25 microns. 7, - Antibiotic according to claim 1 or 2, charac- terized in that the active agent is tenebrimycin VI which has the following properties: tenebrimycin VI is a white solid substance whose titratable radicals have pKa of approx. ron 5.6, 7.2, 8.2 and 9.3 measured by electrometric filtering; in water, tenebrimycin VI has an apparent molecular weight of approximately 396 measured by electrometric titration, tenebrimycin VI has a specific rotation [[alpha]] D25 of +127 (c = 1% w / v. ); Tenebrimycin has the following approximate composition: 'C 44.04%, H 8.40%, N 13.78% and 0 33.62% and exhibits absorption maxima in the infrared (on suspension in mid oil - neral) at 3.05, 3.19, 6.35, 7.47, 8.75, 9.8, 12.3 and 12.95 microns. 8.- Process for the preparation of an antibiotic complex j of tenebrimycins, of tenebrimycin I, I ', II, III, IV, V, VI or of mixtures of such tenebrimycins, characterized in that a Strentomvces tenebrarius is cultured in a medium containing sources of assimilable carbon and nitrogen and mineral salts, submerged in an aerobic medium, until a substantial quantity of the antibiotic is formed by the microorganism in the culture medium . 9. - The method of claim 8, characterized in that the culture medium is maintained at a temperature of about 37 to 43 C and the culture of the microorganism is continued for about 4 to 7 days. 10.- The method of claim 8 or 9, charac- <Desc / Clms Page number 42> ized in that the culture of the microorganism is continued in the absence of visible light. 11 - Process according to claim 8, 9 or 10, characterized in that the tenebrimycin I, tenebrimycin I ', tenebrimycin II, tenebrimycin III, tenebrimycin IV, tenebrimycin V are isolated from the complex of tenebrimycins. , tenebrimycin VI, or mixtures of these tenebrimycins. 12. - Process according to claim 8,9 or 10, characterized in that the Streptomyces tenebrarius is Streptomyces tenebrarius ATCC 17920 or ATCC 17921. 13. A method according to claim 12, characterized in that the Streptomyces tenebrarius ATCC 17920 is cultured, the tenebrimycins complex is isolated from the culture medium and the tenebrimycin II complex is extracted from the tenebrimycin IV, tenebrimycin V or tenebrimycin VI. 14, - Process according to claim 12, characterized in that the Streptomyces tenebrarius ATCC 17921 is cultured and tenebrimycin II is formed containing smaller amounts of tenebrimycin I and of tenebrimycin I '. 15. - A process for preparing the antibiotic according to claim 1, in substance as described with reference to one or other of the examples. 16. Novel antibiotics according to claim 1 substantially as described with particular reference to any of the examples. 17.- Antibiotic obtained by a process according to any one of claims 8 to 15.