CA1046966A - Antibiotic substances and preparation thereof - Google Patents
Antibiotic substances and preparation thereofInfo
- Publication number
- CA1046966A CA1046966A CA234,412A CA234412A CA1046966A CA 1046966 A CA1046966 A CA 1046966A CA 234412 A CA234412 A CA 234412A CA 1046966 A CA1046966 A CA 1046966A
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Abstract
Abstract of the Disclosure A new antibiotic complex, complex 484, components 484-A
and 484-B thereof and a process for their preparation are disclosed. These antibiotic substances are producible by culturing a microorganism of the species Streptomyces in an aqueous nutrient medium under submerged aerobic fermentation conditions. The substances are useful against a broad spectrum of pathogenic bacteria. Methods for their use are disclosed also.
and 484-B thereof and a process for their preparation are disclosed. These antibiotic substances are producible by culturing a microorganism of the species Streptomyces in an aqueous nutrient medium under submerged aerobic fermentation conditions. The substances are useful against a broad spectrum of pathogenic bacteria. Methods for their use are disclosed also.
Description
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6~ , Backqround of the Invention .
a. F d of Invention This invention relates to new antibiotic substances and to a process for producing them. More specifically, this ~invention relates tQ an antibiotic complex designated herein as "complex 484" and to individual antibiotic components thereof.
b. Description of the Prior Art The antibiotic complex of this invention and certain of the components thereof generally resemble those of the streptothricin family of antibiotics but obvious differences do exist as indicated by chromatography and by the products obtained on hydrolysis. More part!cularly, the isolation of the base streptolidine and the amino acTd L-~-lysine following acid hydrolysis of the antibiotic substances !'~
of this invention strongly suggests that the present antibiotic substances are related to the streptothricin family. See for example sfreptothricin, S.A. Waksman and H.B. Woodruff, Proc. Soc. Exptlo Biol.
Med.,49, 207 (1942), streptolin, R.W. Rivett and W.H. Peterson,J. Amer.
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Chem. Soc., 69, 3006 (1947); racemomycins At B, C and D~ H.Taniyama~
; et al., Chem. Pharm. Bull., 19, 1627 (1971) anc fucothricin, Hindustan Antibiot. Bull , 14, 4(1971).- However, the antibiotic substances of this invention can ~bè readily distinguished from the known antibiotics of this class by their different chromatographic behavior and acid hydroly~ ¦
sates, 3S well as spectral and other physical data. Furthermore, the ~ "complex 484"-producing microorganism of this invention appears to be morphologically unique when compared to the known microorganisms , capable of producing streptothricin and strepto,hricin-!ike antibiotics. -~
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Brief_Summary of the Invenlion The antibiotic substances of this invention are obtained by cul~uring a "complex ~8~"-producing organism in an aqueous nutrient medium under submerged aerobic fermentation.
The "complex 484"-producing organism, Streptomyces sp~ NRRL 5490, was obtained from Easter Island soils and samples thereof have been deposi~ed without restrictions with the Northern Utilization and Research Division, Agri-cultural Research Service, U.S. Department of Agriculture, 1~ Peoria, Illinois, U.S.A. ' ;
It is to be understood that the invention is not limited to the use of the particular organism herein described, but includes varTations and mutants obtained by natural selection or by treat-ment of the microorganism with, for instance, ultraviolet rays, -;~ 15 ; X-rays, N-methyl-N'-nitro-N-nitroso-guanîdine, manganese chloride, camphor, nitrogen mustards, and the like.
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~ ~ Streptomyces sp. NRRL 5490 develops abundantly in culture ; media usually employed for cultivation of other organisms of the same genus. It is capable of growing at temperatures ranging from 20 to 30C, preferably at about 25 to 28C, on Czapek's agar, -oatmeal agar, nutrient agar, potato agar, glucose asparagine asar, glycarol asparagine agar, starch agar and peptone beef agar. Also, the organism grows very well on yeast extract agar, malt extract ,~
agar, starch-inorganic salt5 agar, oatmeal-tomato agar and Bennet's ~ .
agar. The aerial mycelium is usually velvety or powdery and gray to pinkTsh gray in color and is spore forming. Sporophores are straight, sometimes spiral-shaped. Mycelial growth is light yellow to almost colorless and in some media pale brown. Occasionally ;
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a yellowish pigrnent is produced. The color of submerge~ mycelium ranges from yellow to rusty brown depending on the medium. The organism is ~12S-negative and melanine-negative, Carbohydrate utilization by StreptomYCes sp. NRRL 5490 ~las studied in carbon utilization agar (ISP Medium 9) according to the procedure standardized by the International Streptomyces Project (ISP).
Utilized carbohydrates included glucose, inositol, fructose, mannitol, rhamnose, xylose, starch and arabinose.
Carbohydrates not utilized were sucrose, raffinose and cellulose.
The environment and nutritional requirements for the fermentation of Streptomyces sp. NRRL 5490 are similar to those ne~essary for the production of antibiotics by other aerobic microorganisms. Thus, aerobiosis is sustained in a liquid nutrient medium inoculated with a sterile culture incubated in flasks placed on shaking machines. For industrial production, metal tanks with internal aeration and agitation by means of paddles can be substituted. The microorganism requires as nutrient elements .
assimTlable carbon and organic nitrogenous substances. The presence of mlneral salts is desirable. Cultlvation is best effected when ` 20 the initial pH o, the culture medium is between 5.5 and 7.5, the optimum pH being~around 5.8 to 6.5.
The utilizable sources of assimilable carbon for the production of the antibiotic substances are very diverse~ there being included sugars (for example, glucose, fructose, mannitol, maltose, arabinose, rhamnose, xylose, and the like, dextrin~ starches of different types of origin, glycerol, inositol and other polyalcohols, and animal and vegetable fats, as well as esters thereof.
The sources of organic assimilable nitrogen ~hich actively stimulate growth and ~avor production of the antibiotic substances of this i invention are substances such as soybean meal, cotton seed meal and other vegetable meals (whole or partially or totally defatted), p ~ v r~ ~
meat flours or animal viscera, various peptones, casein hydrolysates, soybean hydrolysates, yeast hydrolysates, lactalbumin, wheat glutins, distillers solubles, corn steeps, molasses, urea and amino acids.
Mineral salts, such as the chlorides, nitrates, sulfates, carbonates and phosphates of sodium, potassium, ammonium and calcium, should be included in appropriate concentrations. The nutritive medium shouId contain a numbe! of trace elements such as magnesium iron, manganese and zinc:
The fermentation medium is inoculated with a spore suspension of the organism obtained from a fresh slant culture of Streptomyces spO NRRL 5490.
Under the described conditions and with the temperature of cultivation at about 20 - 35C, preferably at about 25 - 28C, substantial production of the desired antibiotic complex is obtained.
Maximum production usually occurs within the period of about twoto nine days.
Thereafter, a variety of procedures may be employed in the isolation and purificatlon of the antibiotic substances, for example, solvent extraction, partition chromatography, chromatography on a variety of absorbents, liquid liquid distribution in a Craig apparatus, and crystallization from solvents.
The antibiotic substances of this invention are harvested in the following general manner:
~ The fermentation mixture is rendered acidic (pH 2 to 6, preferably about 3) by the addition of an acid. The mycelium is separated from the fermentation mixture by conventional means, such as fiItration or centri-fugation. The mycelium free fermentation mixture is adjusted to about j pH 6.0 to 7.0, preferably pH 6.5. The mixture is then treated with an ~-absor~ent preferably by subjecting it to chromatography over a cation exchange resin, preferably one of the Amberlite*lRC-50 type in the *Trade Mark '' ~; .
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ammonium cycle. Other such resins include Zeokar~ 226 and ITke carboxylic acid cation exchange resins. Thereafter, the anti-biotic substances are eluted from the absorbent. In the case where the absorbent is one of the above cation exchange resins, elution is carried out with an alkaline solution, for exaMple, ammonium hydroxide solution, or alkaline buffers, for example, ammonium acetate solutlon. The active fractions contaTning the antibiotTc complex are combined to produce an extract from which the antibiotic complex is isolated. The complex can be further I purified by precipitation from an appropriate solvent system to yield the purified antibiotic complex containing at least three components.
Thereafter, and if desired,the antibi~ot~c complex is separated into various components by known separation techniques.
Partition or absorption chromatography are convenient and effic7ent ~15 techniques for this purpose.
Detai !s of th Invention For m e purpose of this disclosure the term "antibiotic complex" Is used to describe the total antibiotically active material isolable by the process of this invention. This material has been designated as "Complex 484" and its characteristics are described in more detail hereinafter. The term "component" is used herein to describe a factor which is separated or isolated from the complex by known separation techniques. For con-., ~ .
~ venience, individual components are arbitrarily designated 48~-A, 484-B and ~he like. The term "antibiotic substances"
is used herein to describe collectively the antibiotic co~plex and the components.
The antibiotic subs~ances of this invention have useful antimiccobial ac~ivity. For example, they are useful against a ~ 6 ~ *Trade Mark . , .
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broad spectrum o-f pathogenic bacteria.
The antibacterial activity of ~he antibio~ic substances of this invention is demonstrable in standard tests used for this purpose, for example, in the tests described in "Antiseptics, ~isinfectants, Fungicides and Sterilization", G.F. Reddish, Ed., 2nd ed., Lea and Febiger, Philadelphia, 1957 or by D.C. Grove and W.A. Randall in "Assay Methods of Antibiotics", Med. Encycl. Inc., New York 1955.
For example, by employing a test like the serial broth ~0 dilution, see Reddish, cited above, in which dilutions of the I antibiotic substances of this invention in nutrient broth are inoculated with the microorganisms, incubated at 37C for 2 days, respectively9 and examined for the presence of growth, it was show~ that the antibiotic substances inhibit growth totally of a variety of pathogentc microorganisms. The results of such an experiment are reported hereinafter.
When the antibiotic substances of this invention are ~;~
;~ ~ employed as antibacterial agents in warm-blooded animals, e.g. rats, they are used alone or in combination with pharmaceutically accept-able carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard biological practice. For example, an antibacterially effective amount of the substance is administered orally in solid form containing such excipients as starch, sugar, certain types of clay and so forth. Similarly, such an amount can aiso be administered orally in the form of solutions or suspensions, or the antibiotic substance is injected parenterally. For parenteral .
administration the subs-~ance is used in the form of a sterile solution or suspension containing other solutes or suspending agents, for i . , , .
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The dosage of the antibiotic substances will vary with the form of administration and the particular substance chosen. Further-more, it will vary with the particular host under treatment.
Gen0rally, treatment is initiated with small dosages substantially less than the optimum dose of the substance. Thereafter, the dosage is increased by small increments until the optimum effect under ' the circumstances is reached. In general, the antibiotic substances of this inventionare most desirably administered'at a concentration level that will generally afford antibacterially effective results' without causing any harmful or deleterious side effects and preferably at a level that is in a range of from'about 1.0 mg to about 250 mg per kilo per day, although as aforementioned variations will occur.
, :- . -` However, a dosage level that is in the range of from about 10 mg to about 100 ~9 per kilo per day is most desi)rably employed in order ' ~o achieve effective results.
' in addition, the antibiotic substances may be employed topically.
For topical application they are formulated in the ~orm of solutions~ ' creams or lotions in pharmaceutically acceptable vehicles contain-' Ing 0.1 - 5 percent, preferably 2 percent of the antibiotic substances -and are administered topically to the infected area of the skin1 The antibiotic substances can also be used for cleaning and disinfecting laboratory equipment, surgical instruments, locker roomst or sho~er rooms. For such purposes it is preferred to use ~ ~
0.1 - 10% solutions of the substances in water. ¦ "
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In a preferred embodiment of this invenfion the antibiotic substances are obtained in the following manner:
A suitable fermenter is charged with a production medium (see Example 1). After sterilization ~nd coolTng, the medlum is inoculated with a first stage inoculum preparation of StreptomYces sp. N~RL 5490.
The fermentation is allowed to proceed at 20 to 35C, preferably 25 to 28C. During the course of the fermentation the pH of the fermentation mixture is kept between 5.5 to 7.5, preferably ~--5.~ to 6.5 by the addition of a base, for example, 25% sodium, potassium or preferably ammonium hydroxide solution.
After about two to nine days, usually about six days, a maximum titre of the antibiotic substances of this invention is usually obtained. The concentration of the antibiotic substances in the fermentation mixture is readily followed during the course of the fermentation by testing samples of the mixture for their - Jnhibitory effect of a strain of Pseudomonas aeruqinosa or Sarcina lutea as determined by the cup plate method.
~20 Thereafter the fermentation is stopped~ and the pH of the mixtureadJusted to about 3 with an acid, for example, sulfuric or hydrochloric acid. The mycelium is separated from the fermentation mixture by subjecting the mixture to fiItration through a filter aid, for example, diatomaceous earth. The filtrate, i.e. the mycelium-free fermentation mixture,is adjusted to about pH 6.0 to 7.0, preferably -~
6.5, by the addition of base, for example, I N ammonium hydroxide or - sodium hydroxide solution. The adjusted filtrate is now subjected to . .
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~:14~;~6~ii chromatography on a cation exchange resin, for example Amberlite*lRC-50 in the ammonium cycle, in an arnount equal in volums to 1/10 to 1/30 the volum2 of the filtrate. Accordingly, the filtrate is passed through the exchange resin whereby the antibiotic substances are absorbed on the resin. ~he resin is then washed with distilled water and eluted with an alkaline solution preferably ammonium hydroxide (0.25 to I N, p~l = 11.4 to 11.8). The eluate is concentrated, pre~
ferably at reduced pressure at temperatures bet~een 35 to 60G7 The residue is dis~olved in about 50 parts (w/v) of a solvent ; 10 of moderate polarity, for example water, a lower alkanol or mixtures thareof. The solution is treated with decolori7ing charcoal, filtered and the collected charcoal washed with about , 10 to 20 parts of the same solvent. The combined fiItrate and washing are concentrated to about one-tenth of their original volume.
The concentrated solution is treated with about five to ten times its volume of a diluent of low polarity, miscible with the above solvent :.
of moderate polarity,for example, acetone or methyl ethyl ketone in I the case whers water is the above solvent of moderate polarity, or acetone9 methyl ethyl ketone, ether or ethyl acetate in the case where a lower alkanol is the solvent of moderate polarity. The resulting precipitate is collected affording the crude antibiotic complex of this invention. A preferred combination of moderate and ~. .
Iow polarity solvents for the foregoing procedure is a lower alkanol and a lower alkanone, respectively; for example, methanol/acetone, `~ ~ 25 methanol/methyl ethyl ketone, and the like.
Further purification of the crude antibiotic complex is obtained by dissolving and reprecipitating the antibiotic complex from the afore-me~tioned solvent system of moderate and low polarity solvents.
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If desired,-the antibio~ic complex i5 separated into various components. This separation is effected by further subjsction the complex to chroma~ography. A convenient and efficient absorbent for the chromatographic separation is a cross-linked dextran cation exchanger of the CM-Sephadex*type~ preferably CM-Sephadex*C-25. Other suitable absorbents include silica gel, dextran gel, weak cation .-exchange resins, activ~ted carbon, cellulose powder or cellulose based cation exchangers, e.g. carboxymethyl cellulose.
When the CM-Sephadex*is used as the absorbent, stepwise elution of the various antibiotic components is effected by using a volatile buffer9 preferably ammonium acetate or dilute ammonuim hydroxide, and a gradient of pH 7 to about 11.4 for elution.
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By means of the chromatographic separation the antibiotic complex 484 is separated into at least two components which are arbitrarily designated as 484-A and 484-B.
More specifically, the components are readily obtained by subjecting the antibiotic complex to chromatography on silica gel, activated carbon, cellulose powder, weak cation exchange resins, for example Amberlite*lRC-50 (Rohm ~ Haas Co.), cellulose based cation exchangers, for example, carboxymethyl cellulose and cross-iinked dextran based cation exchangers(Sephadex*ion exchangers),for example, CM-Sephade~ ~Pharmacia Co.). The preceding ~ion exchangers can also - be used in a batch treatment. Purification may also be done by gel filtration on dextran gsls such as Sephadex*G-10, G-15 and IH-20 (Pharmacia)or acrylamide gels (Bio-Rad). ~
The ion exchangers are prepared according to the instructions of the manufacturer in water or suitable buffers preferably volatile buffers such as pyridine-acetic acid, ammonium acetate or ammonium formate.
Elution is carried out with dilute acid, dilute alkali or suitable * Trade Mark . . .
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1, buffers, preferably volatile buffers such as ammoniurn acetate employing if necessary a gradient of pH or ionic concentration or a gradient of pH and ionic concentration.
A preferred separation procedure includes chromatography on CM-Sephadex*C-25. The ion exchanger in the NH4~ form is equilibrated to a pH of about 5.0 to 8.0, preferably pH 7.0, using dilute ammonium acetate, preferab!y 0.05N- Elution of the column is initiated using dilute ammonium acetate as the buffer. The pH of the buffer is adjusted with acetic acid or ammonium hydroxide. The material -eluted by buffers up to pH 10.0 is inactive or slightly actlve.
Buffer of pH 10.5 elutes the most polar material with a more ~ .
1 favourable ratio of antibacterial activity to toxicity than the antibiotic complex. This component is arbitrarily assigned the designation 'i: . .
484-A. The latter component is dissolved in aqueous lower alkanol, preferably methanol-water (1:1~ and the solution poured through a column , of a weak base anion exchange resin, for example, Amberlite*lR-45 and -`~ IR-4B, De-Acidite*E, Duolite*A-2 and the like. The eluate is concentrated , to dryness to afford the component substantially free of inorganic ions (i.e.
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~ ammonium and acetate ions). Additional purification of this component, 484-A, is achieved by dissolving and reprecipitating the component in the aforemen-tioned solvent system described for additional purification of the anti-biotic complex. This purtfied component 484-A is more fuliy descr1bed hereinafter.
Having substantially eluted component 484-A as described above, .. ., . . _ ... .
further elution of the column wlth buffer of PH 10.5 and with 0.25 N
; ammonium hydroxide (i.e. an alkaline solution of about pH 11.4) givesmaterial containing, along with a small amount of component 484-A, a less polar fraction as indicated by thin layer chromatography. This material is arbitrarily designated componant 484-B. This latter ... ,,, , :
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component possesses a~higher degree of both antibacterial activity and toxicity than the antibiotic complex ~8~. If desired additional purification of the component is achieved in the same manner as described above for component 484-A. Furthermore, thin layer chromatography on silica gel plates using methànol-chloroform~28%
ammonia (2:1:2) as the mobile phase indicates that component 484-B is a mixture of two or more fractions (Rf's 0.18 and 0.36). This component is described in more detail hereinafter.
Characterization of Antibiotic Com31ex 484 ~o The composition of Complex 484 has indefinite physical constants such as melting point, analytical values and specific rotation because the ratio of various components t~erein varies with the conditions of fermentation and/or . .
extraction of the complex. However, the complex has a specific rotation (~a~D~ of -25 + 3 (methanol), i.e. the specific rotation of several batches ranges from -22 to ~28 (methanol). For example, on batch had a specific rotation, ~]D5 --24.8 (methanol).
Notwithstanding, the complex is characterized readily by its mode of preparation and its subsequent separation into the various components descri~ed hereinO
Additional characterization includes: i (a~ The complex is soluble in water and methanol and sub-stantially insoluble in non polar and moderately non poiar solvents, such as acetone, etherj benzene, ethyl acetate and chloroform ~ i (b) The complex is convertible to acid addition salts, for example, the inorganic acid addition salts, hydrochloride, ; hydrobromide, sulfate and phosphate, and the organic acid addition salts, for example, acetate, pyruvate, tartrate, ... , , ' ~:
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citrate, malate and aspartate.. The salts are readily obtained by treating the complex ~ith the corresponding organic or inorganic acid. The salts possess the same antibacterial activity as the complex itself and are used in the same manner.
(c) Paper chromatography of the complex in the conventional descending manner at 24C for 48 hours on Whatman*No. I paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6:20) gives mainly two areas of antimicrobial activity, as determined by bioautography on agar plates seeded with Sarcina lutea, 25 and 11 cm from the origin.
(d) The complex does not exhibit any characteristic absorption between 220 and 350 nm in its ultraviolet absorption spectrum (95% ethanol).
. (e) A representative infrared absorption spectrum of the complex in KBr pellet is reproduced in accompanying Figure I and shows characteristic absorption bands at 3330, 2925, 2875 (sh), 1710, 1650, 1560 ~sh), 1450, .1380, 1170, 1080, 1030, 970, 920 and _ I
820 cm .
:~ (f) The minimum inhibitory concentration of the antibiotic complex against various microorganisms are listed in Table I below.
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(9~ The complex exhibits a lD5~ (i.p., rnice) ranging from 350 to 450 mg/kg and a LDo (P--~ mice) ot > 1~0 g/kg.
In protection studies mice were infected with E. co ATCC 10536. At one hour and again at five hours after infection, mice were administered 20 mg/kg (s.c.) of the antibiotic complex 484. At thTs dose 50% of these mice were protected. At 25 mg/kg all the mice were protected.
Furthermore, the complex gives a positive reaction to ninhydrin, a yellow color with Elson-Morgan reagent ti.e. a negative reaction), a negative reaction with Sakaguchi reagent and a positive reaction with silver nitrate reagent.
Characterization of Antibiotic Component 484-A
Component 484-A is characterized by its mode of preparation and the following additional characterizat7On: .
a) Component 484-A, similar to the complex ~84, is soluble in -.
~ water and methanol and substantially insoluble in acetone, - ether, benzene, ethyl acetate and chloroform.
b) Component 484-A is convertible to acid addition salts, for example the inorganic acid addition saits, hydrochloride, hydrobromide~ sulfate and phosphate, and the organic acid addition salts, for example, acetate, pyruvate, tartrate, cttrate, malate and aspartate. The salts are readily obtained by treating the component with the corresponding organic or inorganic acid~ The salts possess the same antibacterial activity as the component itself and are used in the same manner.
c) Paper chromatography of component 484-A in the conventional descending manner at 24C for 48 hours on Whatman*No. I
paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30 20:6:20)gives one main zone of antimicrobial activity (23 - 25 cm. from origin) as shown by bioautography on plates seeded with Sarcina lutea. When the duplicate paper chromatograms are - .. .. ~
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-i6 sprayed with ninhydrin 1wo main spots (13 - 15 crn and 23 - 25 cm from origin) are detected. The s~lfate salt of component 434-A
under the above cond7tions ofdescending chromatography and bioauto-graphy shows one bioactive zone abou, 18 to 20 cm from origin. Ninhydrin spray reveals two spots ~10 cm and 18 - 20 cm from origin). Racemomycin A sulfate, H. Taniyama and F. Miyoshi, Chem. Pharm. Bull. (Tokyo), 10, 156 (1962), a known streptothricin-like antibiotic used as a standard,shows a wide zone of inhibition 21 - 26 cm. from origin when tested under the same 0 conditions and a duplicate chromatogram sprayed with ninhydride reveals a spot at 24 cm.
d) Paper chromatography of the sulfate salt of component 484-A in the conventional ascending manner at 24C
for 16 hours on Whatman*No~ I paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20 6:24) gives two main spots as detected by ninhydrin Rf 0.15 - 0.16 and Rf 0.22 - 0.24; Racemomycin A sulfate ,! gives a spot with Rf 0.23 - 0.24 under these conditions.
,~ e) Circular paper chromatography [see, M.l. Horowitz20~ and C.P. Schaffner, Anal. Chem., 30 1616 (1958)],of the sulfate , ~ , : -.
salt of component 484-A using Whatman*No. I paper and a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6 24) gives one zone of antimicrqbial activity at Rf 0.40 -0.42 and two zones with Rf 0.30 - 0.32 and Rf 0.40 - 0.42 as~detected . ~ e ' by ninhydrin spray; racemomycin A sulfate gives a spot with Rf 0.44 -0.46 in this system as detected by ninhydrin spray. - ~
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f) A representative infrared absorption spectrum o~ component 484-A in KBr pellet is reprcd~ced in the accompanyina Figure 2 and shows characteristic absorption bands at 3300, 3080 (sh), 2940 (sh), 1710 (sh), 1660, 1550, 1390, 1350, 1310, 1105, 955, 920 and 850 cm~l.
g) The minimum inhibitory concentration of component 484-A
against various microorganisms is listed in Table 1, see below.
h) Component 484-A exhibits a LD50 (i.P.J ~ice) >1.0 g/kg.
This LD50 readily distinguishes this substance from other known streptothricin-like antibiotics.
i~ AmTno acid analysis of component 484-A indicated the presence of a streptolidine moiety and a L-~-lysine moiety in about a I to I ra~ion.
In protection studies mice were infected with ATCC 10536. At one hour and again at five hours a~ter infection, mice were given ~5 mg/kg, (sOc) of component 484-A. The result was complete protection of the mice. ~ -Furthermore, like the complex, component 484-A gives - positive reactions with ninhydrin and siIver nitrate, and negative reactions wi1h Elson-Morgan and Sakaguchi reagents.
The specific rotation ([~]D5) of component 484-A is -25 + 3~
(H20)~ i.e. the specific rotation of several batches range from -22 to -28 (H20). One sample of component 484-A had the specific rotation [~]~5 = -27C (HzO~.
The specific rotation ([a]D25) of several batches of component 484-A ranges from -22 to -28 ~H20). One sample of component 484-A had the specific rotation [a]D = -27C (H20).
Characterizatlon of Antibiotic Component 484-B
Componenf 484-B is characterized by its mode of preparation and the following additional characterization:
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a) Component 484-B; like the complex 484 and the component 484-A, is soluble in water and methanol and substantially insoluble in acetone, ether, benzene, ethyl acetate and chloroform.
b) Component 484-B is convertible to acid addition salts, for example, the inorganic acid addition salts, hydrochlorTde, hydro- ' bromide, sulfate and phosphate, and the organic acid addition salts, fo'r' example`~' a'cetate, pyruvate, tartrate, citrate, malate and aspartate. The salts are readily obtained by treatment of the componenJ with the corresponding organic or inorganic acid.
The salts possess the same antibacterial activity as the component itself and are used in the same manner. -~
c) Paper chromatography of the component 484-B in the conventional descending manner at 24C for 48 hours on Whatman -No. I paper using a solvent system consisting of n-propanol, ' -' ' pyridine, acetic acid and water (6:4:1.5:4) gives a main area of antimicrobial activity, as determined by bioautography on ~gar plates seeded with ~ ~ at 17 cm and minor zones at 24 and 8 cm from the origin.
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d) Circuiar chromatography utilizing Whatman*No. l paper and~ a solv0nt system consisting of n-propanol, pyridine, acetic acid and water (30:20:6~24) gives three zones ' ;~ as detected by ninhydrin (Rf 0.16, 0.29'and 0.42).
e) A representative infrared absorption spectrum of component ' 4B4-B is reproduced in accompanying Figure 3 and shows characteristic 1 bands at 3280, 3070, 2930, 1710, 1655, 1550, 1395, 1075 and 920 crn 1.
f) The minimum inhibitory concentration of component 484-B
` against various microorganisms is listed in Table 1, see below.
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g) Component 484-B exhibits a LD50 (i P ~ mice) of about 100 mg/kg. - -* Trade Name ,, . ~ , - :
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h) Amino acid an~lysis of component 484-B indicates the presence of a streptolidi.ne moiety and a L-~-lysine rnoiety in about a I to 2 ratio.
The specific rotation ([a~2D5) of this compo~nent is -12 + 2 (H20), i.e. the specific rotation of several batches of component 484-A ranged from -10 to -14 (H20). One sampie of component 484-B had a specific rotation, ~a~D4 = -12 (H20).
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TABLE I
Minimum Inhibitory Concentration (MIC) in mcq/ml for the Antibiotic Substances of this Invention _ _ ~est;MicroorqanismComplex 484 Component 484-A Component 484-B
Staphylococcus pyogenes(penicillin- 0-4 0.8 ~D.I
sens i t i ve ) , Staphylococcus , 'rYes`istant~P i 3.2 0.4 : -Streptococcus -faecalls 50.0 50.0 12.5 - Escherichia coli 3.2 3.2 0.8 Aerobacter aerogenes I o6 1.6 0.4.
, _ , _ . . .
Salmonalla pullorum 50.0 . 50-0 6.4 .
Pseudomonas aeruginosa 50~,0 50.0 12.5 , Proteus mirabilis3.2 3.2 0.8 Proteus vulgaris 1.6 3.2 0.4 : Klebsiella pneumoniae 1.6 1.6 0.4 Serratia ~,arcescens 3.2 3.2 0.8 ' ~, The following examples illustrate ~urther this invention.
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EXhMPlE I
_reparation and Purificatlon of ~ x 48 -M roorqanism StreDtomvces sp. NRRL 5490 was grown and maintained on oatmeal-tomato paste agar slants (T.G. Pridham, et alO, Antibiotic Annual 1956 - 1957, Medical Encyclopedia Inc., ; New York, p. 947) and in Roux bottles containjng the same medium. Good growth was obtained after 7 days of incubation at 28C. Spores from one Roux bottle were washed off and suspended into 50 ml of sterile distilled water. This suspension was used to inoculate the first stage inoculum.
Flrst Staae Inoculum The first stage inoculum medium is an aqueous medium having the following constituents glucose (Cerelose)* 2.0%
` ~ peptone (Neopeptone)* 0.5%
tomato paste 1.0%
corn meal 0.8%
"Blackstrap" molasses 2.0%
sodium chloride 0.3%
pH adjusted to 7.8 withammonium hy~roxide This first stage inoculum medium (3.2 1), contained in a twenty-four litre Florence flask,was sterilized at 121C for I hour, cooied and inoculated with (32 ml, 1% inoculum) of the spore suspension described above. The inoculated flask was incubated for 30 hours at 28C on a reciprocating shaker operating at 65 rev/min t4"-stroke).
Production _t~e ` The production stage was run in 250 liter New Brunswlck fermenters, tModel F-250 made by the New Brunswick Scientific Co., New Brunswick, N.~.~, e~uipped with automatic antifoam addition system and pH recorder-controller. The fermenters were charged ~i - 2:l , *Trade Mark ' ~' '' ' ' ', :. ' .' -'. ' ' :
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with 160 liters of the same medium as described abové, and lard oil (0.1% v/v, Larex*No. i, Swift Canadian Co., Toronto)~/as added as an antifoam agent before sterilization. The fermenters were sterilized at 121C for 35 minutes. The sterilized and cooled fermenters were inoculated with one flask (3.2 1, 2% inoculum) of first stage inoculum. Incubation temperature 28C;
aeration: 0.5 vol/vol/min; agitation: 250 rev/min. The antifoam agent, used on demand, was Mazur*DF-143PX (Mazur Chemical Corp., Gurnee, Illinois). During the course of fermentation pH was kept at or above 6.0 by automatic addition of 25% sod1um hydroxide solution~
After six days incubation the pH of the fermentation broth reached 7.5 and a 1.3 cm diameter fiIter paper disc in broth gave an inhibition zone of 23 mm on an agar plate seeded :
with a strain of Pseudomonas aeruainosa. At this time the ; packed cell volume (PCV) was 24%. The fermentation was stopped, the pH of the broth was adjusted to 3.0 with sulfuric acid, and ,~ .
diatomaceous earth ~5% v/v) was added. The mixture was subjected to filtration. The filtrate containing the antibiotic principle was retained and the mycelium discarded.
Extraction and Recovery The pH of the fiItrate (volume = 150 liters) was adjusted to 6.5 with I N ammonium hydroxide solution. The filtrate was passed through a coiumn containing 7 liters of Amberlite*lRC 50 NH
resin at a rate of 20 liters per hour. The product was absorbed on the resin and the eluate was rejected. The resin was washed with deionized water until the washings were colorless. The product *Trade Mark ,,~,~j;.~
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was eluted from the resin with 12 liters of I N ammonium hydroxide solution. The ammoniurn hydroxide eluate was evaporated to dryness under reduced pressure at 40C. The residue was dissolved in 3 liters of methanol with vigorous stirring. The solution was filtered. The fi~trate containing the active principle was de-colorized by stirr7ng for 20 min. witn 1~ charcoal (Darc~ G-60) and filtered. The char,coal was washed with I liter of methanol with stirring. The methanol fiItrate and washings were evaporated to 500 ml under reduced pressure. Addition of seven volumes of acetone to the methanol concentrate precipitated the antibiotic complex.
Further purification was obtained by dissolving the ~ -latter product (40 9) in 400 ml~ of methanol and slowiy adding 2800 ml of acetone to the vigorously stirred solution. The resulting precipitate was collected. R2petition of tnis , 15 purification step gave the purifTed complex.
.
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6~;6 Se~ _ation of the Complex 4~4 into Com~onents 484-A and 484-B.
CM-Sephadex*C-25 in the N~4+ form was equilibrated with 0.05 N ammonium acetate (pH 7). The complex (10.0 9), obtained as described in Example 1, was dissoived in a small volume of water and the pH adjusted to 7 with aceti¢ acid. This solution was applied to a column containing 300 ml of the above ion exchanger, CM-Sephade~C-25. The column was eluted stepwise with buffered O.05 M ammonium acetate of increasing pH, the pH being adjusted by the addition of ammonium hydroxide or acetic acid. The eluted fractions were evaporated to dryness under reduced pressure. Accordingly, the column was eluted first with 2 - 3 bed volumes of ammonium acetate buffer (pH 7), followed by 2 - 3 bed volumes of ammonium acetate buffer (pH 9.0). These ' 15 fractions were inactlve and were discarded. The column was eluted next with 3 - 4 bed volumes of ammonium acetate butfer (pH 10). The material obtained had a low order of activity.
Thereafter the column was eluted with 3 - 4 bed volumes of ammonium acetate buffer (pH 10.5). The first I - 1.5 bed volumes of eluate contained material of iow activity and the next I - 2 bed volumes contalned material of high activity. This material was , designated component 484-A.
. ~ , Further elution of the column with the same buffer (3rd and 4th bed volumes), foliowed by elution with 2 - 3 bed volumss of 0.25 N ammonium hydroxide gave material of high activity. ~his material was designated component 484-B .
Component 484-A or 484-B were purified further by dissolving each component in a small volume of 50% methanol-water and passing the solution through a column ofAmberlite*lR~45 (OH-). The .... .
6~ , Backqround of the Invention .
a. F d of Invention This invention relates to new antibiotic substances and to a process for producing them. More specifically, this ~invention relates tQ an antibiotic complex designated herein as "complex 484" and to individual antibiotic components thereof.
b. Description of the Prior Art The antibiotic complex of this invention and certain of the components thereof generally resemble those of the streptothricin family of antibiotics but obvious differences do exist as indicated by chromatography and by the products obtained on hydrolysis. More part!cularly, the isolation of the base streptolidine and the amino acTd L-~-lysine following acid hydrolysis of the antibiotic substances !'~
of this invention strongly suggests that the present antibiotic substances are related to the streptothricin family. See for example sfreptothricin, S.A. Waksman and H.B. Woodruff, Proc. Soc. Exptlo Biol.
Med.,49, 207 (1942), streptolin, R.W. Rivett and W.H. Peterson,J. Amer.
, :
Chem. Soc., 69, 3006 (1947); racemomycins At B, C and D~ H.Taniyama~
; et al., Chem. Pharm. Bull., 19, 1627 (1971) anc fucothricin, Hindustan Antibiot. Bull , 14, 4(1971).- However, the antibiotic substances of this invention can ~bè readily distinguished from the known antibiotics of this class by their different chromatographic behavior and acid hydroly~ ¦
sates, 3S well as spectral and other physical data. Furthermore, the ~ "complex 484"-producing microorganism of this invention appears to be morphologically unique when compared to the known microorganisms , capable of producing streptothricin and strepto,hricin-!ike antibiotics. -~
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Brief_Summary of the Invenlion The antibiotic substances of this invention are obtained by cul~uring a "complex ~8~"-producing organism in an aqueous nutrient medium under submerged aerobic fermentation.
The "complex 484"-producing organism, Streptomyces sp~ NRRL 5490, was obtained from Easter Island soils and samples thereof have been deposi~ed without restrictions with the Northern Utilization and Research Division, Agri-cultural Research Service, U.S. Department of Agriculture, 1~ Peoria, Illinois, U.S.A. ' ;
It is to be understood that the invention is not limited to the use of the particular organism herein described, but includes varTations and mutants obtained by natural selection or by treat-ment of the microorganism with, for instance, ultraviolet rays, -;~ 15 ; X-rays, N-methyl-N'-nitro-N-nitroso-guanîdine, manganese chloride, camphor, nitrogen mustards, and the like.
:' ' ;
~ ~ Streptomyces sp. NRRL 5490 develops abundantly in culture ; media usually employed for cultivation of other organisms of the same genus. It is capable of growing at temperatures ranging from 20 to 30C, preferably at about 25 to 28C, on Czapek's agar, -oatmeal agar, nutrient agar, potato agar, glucose asparagine asar, glycarol asparagine agar, starch agar and peptone beef agar. Also, the organism grows very well on yeast extract agar, malt extract ,~
agar, starch-inorganic salt5 agar, oatmeal-tomato agar and Bennet's ~ .
agar. The aerial mycelium is usually velvety or powdery and gray to pinkTsh gray in color and is spore forming. Sporophores are straight, sometimes spiral-shaped. Mycelial growth is light yellow to almost colorless and in some media pale brown. Occasionally ;
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a yellowish pigrnent is produced. The color of submerge~ mycelium ranges from yellow to rusty brown depending on the medium. The organism is ~12S-negative and melanine-negative, Carbohydrate utilization by StreptomYCes sp. NRRL 5490 ~las studied in carbon utilization agar (ISP Medium 9) according to the procedure standardized by the International Streptomyces Project (ISP).
Utilized carbohydrates included glucose, inositol, fructose, mannitol, rhamnose, xylose, starch and arabinose.
Carbohydrates not utilized were sucrose, raffinose and cellulose.
The environment and nutritional requirements for the fermentation of Streptomyces sp. NRRL 5490 are similar to those ne~essary for the production of antibiotics by other aerobic microorganisms. Thus, aerobiosis is sustained in a liquid nutrient medium inoculated with a sterile culture incubated in flasks placed on shaking machines. For industrial production, metal tanks with internal aeration and agitation by means of paddles can be substituted. The microorganism requires as nutrient elements .
assimTlable carbon and organic nitrogenous substances. The presence of mlneral salts is desirable. Cultlvation is best effected when ` 20 the initial pH o, the culture medium is between 5.5 and 7.5, the optimum pH being~around 5.8 to 6.5.
The utilizable sources of assimilable carbon for the production of the antibiotic substances are very diverse~ there being included sugars (for example, glucose, fructose, mannitol, maltose, arabinose, rhamnose, xylose, and the like, dextrin~ starches of different types of origin, glycerol, inositol and other polyalcohols, and animal and vegetable fats, as well as esters thereof.
The sources of organic assimilable nitrogen ~hich actively stimulate growth and ~avor production of the antibiotic substances of this i invention are substances such as soybean meal, cotton seed meal and other vegetable meals (whole or partially or totally defatted), p ~ v r~ ~
meat flours or animal viscera, various peptones, casein hydrolysates, soybean hydrolysates, yeast hydrolysates, lactalbumin, wheat glutins, distillers solubles, corn steeps, molasses, urea and amino acids.
Mineral salts, such as the chlorides, nitrates, sulfates, carbonates and phosphates of sodium, potassium, ammonium and calcium, should be included in appropriate concentrations. The nutritive medium shouId contain a numbe! of trace elements such as magnesium iron, manganese and zinc:
The fermentation medium is inoculated with a spore suspension of the organism obtained from a fresh slant culture of Streptomyces spO NRRL 5490.
Under the described conditions and with the temperature of cultivation at about 20 - 35C, preferably at about 25 - 28C, substantial production of the desired antibiotic complex is obtained.
Maximum production usually occurs within the period of about twoto nine days.
Thereafter, a variety of procedures may be employed in the isolation and purificatlon of the antibiotic substances, for example, solvent extraction, partition chromatography, chromatography on a variety of absorbents, liquid liquid distribution in a Craig apparatus, and crystallization from solvents.
The antibiotic substances of this invention are harvested in the following general manner:
~ The fermentation mixture is rendered acidic (pH 2 to 6, preferably about 3) by the addition of an acid. The mycelium is separated from the fermentation mixture by conventional means, such as fiItration or centri-fugation. The mycelium free fermentation mixture is adjusted to about j pH 6.0 to 7.0, preferably pH 6.5. The mixture is then treated with an ~-absor~ent preferably by subjecting it to chromatography over a cation exchange resin, preferably one of the Amberlite*lRC-50 type in the *Trade Mark '' ~; .
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ammonium cycle. Other such resins include Zeokar~ 226 and ITke carboxylic acid cation exchange resins. Thereafter, the anti-biotic substances are eluted from the absorbent. In the case where the absorbent is one of the above cation exchange resins, elution is carried out with an alkaline solution, for exaMple, ammonium hydroxide solution, or alkaline buffers, for example, ammonium acetate solutlon. The active fractions contaTning the antibiotTc complex are combined to produce an extract from which the antibiotic complex is isolated. The complex can be further I purified by precipitation from an appropriate solvent system to yield the purified antibiotic complex containing at least three components.
Thereafter, and if desired,the antibi~ot~c complex is separated into various components by known separation techniques.
Partition or absorption chromatography are convenient and effic7ent ~15 techniques for this purpose.
Detai !s of th Invention For m e purpose of this disclosure the term "antibiotic complex" Is used to describe the total antibiotically active material isolable by the process of this invention. This material has been designated as "Complex 484" and its characteristics are described in more detail hereinafter. The term "component" is used herein to describe a factor which is separated or isolated from the complex by known separation techniques. For con-., ~ .
~ venience, individual components are arbitrarily designated 48~-A, 484-B and ~he like. The term "antibiotic substances"
is used herein to describe collectively the antibiotic co~plex and the components.
The antibiotic subs~ances of this invention have useful antimiccobial ac~ivity. For example, they are useful against a ~ 6 ~ *Trade Mark . , .
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broad spectrum o-f pathogenic bacteria.
The antibacterial activity of ~he antibio~ic substances of this invention is demonstrable in standard tests used for this purpose, for example, in the tests described in "Antiseptics, ~isinfectants, Fungicides and Sterilization", G.F. Reddish, Ed., 2nd ed., Lea and Febiger, Philadelphia, 1957 or by D.C. Grove and W.A. Randall in "Assay Methods of Antibiotics", Med. Encycl. Inc., New York 1955.
For example, by employing a test like the serial broth ~0 dilution, see Reddish, cited above, in which dilutions of the I antibiotic substances of this invention in nutrient broth are inoculated with the microorganisms, incubated at 37C for 2 days, respectively9 and examined for the presence of growth, it was show~ that the antibiotic substances inhibit growth totally of a variety of pathogentc microorganisms. The results of such an experiment are reported hereinafter.
When the antibiotic substances of this invention are ~;~
;~ ~ employed as antibacterial agents in warm-blooded animals, e.g. rats, they are used alone or in combination with pharmaceutically accept-able carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard biological practice. For example, an antibacterially effective amount of the substance is administered orally in solid form containing such excipients as starch, sugar, certain types of clay and so forth. Similarly, such an amount can aiso be administered orally in the form of solutions or suspensions, or the antibiotic substance is injected parenterally. For parenteral .
administration the subs-~ance is used in the form of a sterile solution or suspension containing other solutes or suspending agents, for i . , , .
A~IP-6054-1-CI
. ~' , ;96~6 example, enouyh saline or glucose to make the solu-tion isotonic, bile salts, acacia, gela~in, sorbitan monoleate, polysorbate ~0 (oleate esters of sorbitol and its anhydrides copolymerized ~,Jith '' ethylene oxide) and the like.
The dosage of the antibiotic substances will vary with the form of administration and the particular substance chosen. Further-more, it will vary with the particular host under treatment.
Gen0rally, treatment is initiated with small dosages substantially less than the optimum dose of the substance. Thereafter, the dosage is increased by small increments until the optimum effect under ' the circumstances is reached. In general, the antibiotic substances of this inventionare most desirably administered'at a concentration level that will generally afford antibacterially effective results' without causing any harmful or deleterious side effects and preferably at a level that is in a range of from'about 1.0 mg to about 250 mg per kilo per day, although as aforementioned variations will occur.
, :- . -` However, a dosage level that is in the range of from about 10 mg to about 100 ~9 per kilo per day is most desi)rably employed in order ' ~o achieve effective results.
' in addition, the antibiotic substances may be employed topically.
For topical application they are formulated in the ~orm of solutions~ ' creams or lotions in pharmaceutically acceptable vehicles contain-' Ing 0.1 - 5 percent, preferably 2 percent of the antibiotic substances -and are administered topically to the infected area of the skin1 The antibiotic substances can also be used for cleaning and disinfecting laboratory equipment, surgical instruments, locker roomst or sho~er rooms. For such purposes it is preferred to use ~ ~
0.1 - 10% solutions of the substances in water. ¦ "
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In a preferred embodiment of this invenfion the antibiotic substances are obtained in the following manner:
A suitable fermenter is charged with a production medium (see Example 1). After sterilization ~nd coolTng, the medlum is inoculated with a first stage inoculum preparation of StreptomYces sp. N~RL 5490.
The fermentation is allowed to proceed at 20 to 35C, preferably 25 to 28C. During the course of the fermentation the pH of the fermentation mixture is kept between 5.5 to 7.5, preferably ~--5.~ to 6.5 by the addition of a base, for example, 25% sodium, potassium or preferably ammonium hydroxide solution.
After about two to nine days, usually about six days, a maximum titre of the antibiotic substances of this invention is usually obtained. The concentration of the antibiotic substances in the fermentation mixture is readily followed during the course of the fermentation by testing samples of the mixture for their - Jnhibitory effect of a strain of Pseudomonas aeruqinosa or Sarcina lutea as determined by the cup plate method.
~20 Thereafter the fermentation is stopped~ and the pH of the mixtureadJusted to about 3 with an acid, for example, sulfuric or hydrochloric acid. The mycelium is separated from the fermentation mixture by subjecting the mixture to fiItration through a filter aid, for example, diatomaceous earth. The filtrate, i.e. the mycelium-free fermentation mixture,is adjusted to about pH 6.0 to 7.0, preferably -~
6.5, by the addition of base, for example, I N ammonium hydroxide or - sodium hydroxide solution. The adjusted filtrate is now subjected to . .
.
I~-IP-~0~4-l-f~l ,, .
~:14~;~6~ii chromatography on a cation exchange resin, for example Amberlite*lRC-50 in the ammonium cycle, in an arnount equal in volums to 1/10 to 1/30 the volum2 of the filtrate. Accordingly, the filtrate is passed through the exchange resin whereby the antibiotic substances are absorbed on the resin. ~he resin is then washed with distilled water and eluted with an alkaline solution preferably ammonium hydroxide (0.25 to I N, p~l = 11.4 to 11.8). The eluate is concentrated, pre~
ferably at reduced pressure at temperatures bet~een 35 to 60G7 The residue is dis~olved in about 50 parts (w/v) of a solvent ; 10 of moderate polarity, for example water, a lower alkanol or mixtures thareof. The solution is treated with decolori7ing charcoal, filtered and the collected charcoal washed with about , 10 to 20 parts of the same solvent. The combined fiItrate and washing are concentrated to about one-tenth of their original volume.
The concentrated solution is treated with about five to ten times its volume of a diluent of low polarity, miscible with the above solvent :.
of moderate polarity,for example, acetone or methyl ethyl ketone in I the case whers water is the above solvent of moderate polarity, or acetone9 methyl ethyl ketone, ether or ethyl acetate in the case where a lower alkanol is the solvent of moderate polarity. The resulting precipitate is collected affording the crude antibiotic complex of this invention. A preferred combination of moderate and ~. .
Iow polarity solvents for the foregoing procedure is a lower alkanol and a lower alkanone, respectively; for example, methanol/acetone, `~ ~ 25 methanol/methyl ethyl ketone, and the like.
Further purification of the crude antibiotic complex is obtained by dissolving and reprecipitating the antibiotic complex from the afore-me~tioned solvent system of moderate and low polarity solvents.
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If desired,-the antibio~ic complex i5 separated into various components. This separation is effected by further subjsction the complex to chroma~ography. A convenient and efficient absorbent for the chromatographic separation is a cross-linked dextran cation exchanger of the CM-Sephadex*type~ preferably CM-Sephadex*C-25. Other suitable absorbents include silica gel, dextran gel, weak cation .-exchange resins, activ~ted carbon, cellulose powder or cellulose based cation exchangers, e.g. carboxymethyl cellulose.
When the CM-Sephadex*is used as the absorbent, stepwise elution of the various antibiotic components is effected by using a volatile buffer9 preferably ammonium acetate or dilute ammonuim hydroxide, and a gradient of pH 7 to about 11.4 for elution.
. .
By means of the chromatographic separation the antibiotic complex 484 is separated into at least two components which are arbitrarily designated as 484-A and 484-B.
More specifically, the components are readily obtained by subjecting the antibiotic complex to chromatography on silica gel, activated carbon, cellulose powder, weak cation exchange resins, for example Amberlite*lRC-50 (Rohm ~ Haas Co.), cellulose based cation exchangers, for example, carboxymethyl cellulose and cross-iinked dextran based cation exchangers(Sephadex*ion exchangers),for example, CM-Sephade~ ~Pharmacia Co.). The preceding ~ion exchangers can also - be used in a batch treatment. Purification may also be done by gel filtration on dextran gsls such as Sephadex*G-10, G-15 and IH-20 (Pharmacia)or acrylamide gels (Bio-Rad). ~
The ion exchangers are prepared according to the instructions of the manufacturer in water or suitable buffers preferably volatile buffers such as pyridine-acetic acid, ammonium acetate or ammonium formate.
Elution is carried out with dilute acid, dilute alkali or suitable * Trade Mark . . .
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1, buffers, preferably volatile buffers such as ammoniurn acetate employing if necessary a gradient of pH or ionic concentration or a gradient of pH and ionic concentration.
A preferred separation procedure includes chromatography on CM-Sephadex*C-25. The ion exchanger in the NH4~ form is equilibrated to a pH of about 5.0 to 8.0, preferably pH 7.0, using dilute ammonium acetate, preferab!y 0.05N- Elution of the column is initiated using dilute ammonium acetate as the buffer. The pH of the buffer is adjusted with acetic acid or ammonium hydroxide. The material -eluted by buffers up to pH 10.0 is inactive or slightly actlve.
Buffer of pH 10.5 elutes the most polar material with a more ~ .
1 favourable ratio of antibacterial activity to toxicity than the antibiotic complex. This component is arbitrarily assigned the designation 'i: . .
484-A. The latter component is dissolved in aqueous lower alkanol, preferably methanol-water (1:1~ and the solution poured through a column , of a weak base anion exchange resin, for example, Amberlite*lR-45 and -`~ IR-4B, De-Acidite*E, Duolite*A-2 and the like. The eluate is concentrated , to dryness to afford the component substantially free of inorganic ions (i.e.
. . .
~ ammonium and acetate ions). Additional purification of this component, 484-A, is achieved by dissolving and reprecipitating the component in the aforemen-tioned solvent system described for additional purification of the anti-biotic complex. This purtfied component 484-A is more fuliy descr1bed hereinafter.
Having substantially eluted component 484-A as described above, .. ., . . _ ... .
further elution of the column wlth buffer of PH 10.5 and with 0.25 N
; ammonium hydroxide (i.e. an alkaline solution of about pH 11.4) givesmaterial containing, along with a small amount of component 484-A, a less polar fraction as indicated by thin layer chromatography. This material is arbitrarily designated componant 484-B. This latter ... ,,, , :
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component possesses a~higher degree of both antibacterial activity and toxicity than the antibiotic complex ~8~. If desired additional purification of the component is achieved in the same manner as described above for component 484-A. Furthermore, thin layer chromatography on silica gel plates using methànol-chloroform~28%
ammonia (2:1:2) as the mobile phase indicates that component 484-B is a mixture of two or more fractions (Rf's 0.18 and 0.36). This component is described in more detail hereinafter.
Characterization of Antibiotic Com31ex 484 ~o The composition of Complex 484 has indefinite physical constants such as melting point, analytical values and specific rotation because the ratio of various components t~erein varies with the conditions of fermentation and/or . .
extraction of the complex. However, the complex has a specific rotation (~a~D~ of -25 + 3 (methanol), i.e. the specific rotation of several batches ranges from -22 to ~28 (methanol). For example, on batch had a specific rotation, ~]D5 --24.8 (methanol).
Notwithstanding, the complex is characterized readily by its mode of preparation and its subsequent separation into the various components descri~ed hereinO
Additional characterization includes: i (a~ The complex is soluble in water and methanol and sub-stantially insoluble in non polar and moderately non poiar solvents, such as acetone, etherj benzene, ethyl acetate and chloroform ~ i (b) The complex is convertible to acid addition salts, for example, the inorganic acid addition salts, hydrochloride, ; hydrobromide, sulfate and phosphate, and the organic acid addition salts, for example, acetate, pyruvate, tartrate, ... , , ' ~:
I
.
citrate, malate and aspartate.. The salts are readily obtained by treating the complex ~ith the corresponding organic or inorganic acid. The salts possess the same antibacterial activity as the complex itself and are used in the same manner.
(c) Paper chromatography of the complex in the conventional descending manner at 24C for 48 hours on Whatman*No. I paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6:20) gives mainly two areas of antimicrobial activity, as determined by bioautography on agar plates seeded with Sarcina lutea, 25 and 11 cm from the origin.
(d) The complex does not exhibit any characteristic absorption between 220 and 350 nm in its ultraviolet absorption spectrum (95% ethanol).
. (e) A representative infrared absorption spectrum of the complex in KBr pellet is reproduced in accompanying Figure I and shows characteristic absorption bands at 3330, 2925, 2875 (sh), 1710, 1650, 1560 ~sh), 1450, .1380, 1170, 1080, 1030, 970, 920 and _ I
820 cm .
:~ (f) The minimum inhibitory concentration of the antibiotic complex against various microorganisms are listed in Table I below.
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(9~ The complex exhibits a lD5~ (i.p., rnice) ranging from 350 to 450 mg/kg and a LDo (P--~ mice) ot > 1~0 g/kg.
In protection studies mice were infected with E. co ATCC 10536. At one hour and again at five hours after infection, mice were administered 20 mg/kg (s.c.) of the antibiotic complex 484. At thTs dose 50% of these mice were protected. At 25 mg/kg all the mice were protected.
Furthermore, the complex gives a positive reaction to ninhydrin, a yellow color with Elson-Morgan reagent ti.e. a negative reaction), a negative reaction with Sakaguchi reagent and a positive reaction with silver nitrate reagent.
Characterization of Antibiotic Component 484-A
Component 484-A is characterized by its mode of preparation and the following additional characterizat7On: .
a) Component 484-A, similar to the complex ~84, is soluble in -.
~ water and methanol and substantially insoluble in acetone, - ether, benzene, ethyl acetate and chloroform.
b) Component 484-A is convertible to acid addition salts, for example the inorganic acid addition saits, hydrochloride, hydrobromide~ sulfate and phosphate, and the organic acid addition salts, for example, acetate, pyruvate, tartrate, cttrate, malate and aspartate. The salts are readily obtained by treating the component with the corresponding organic or inorganic acid~ The salts possess the same antibacterial activity as the component itself and are used in the same manner.
c) Paper chromatography of component 484-A in the conventional descending manner at 24C for 48 hours on Whatman*No. I
paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30 20:6:20)gives one main zone of antimicrobial activity (23 - 25 cm. from origin) as shown by bioautography on plates seeded with Sarcina lutea. When the duplicate paper chromatograms are - .. .. ~
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-i6 sprayed with ninhydrin 1wo main spots (13 - 15 crn and 23 - 25 cm from origin) are detected. The s~lfate salt of component 434-A
under the above cond7tions ofdescending chromatography and bioauto-graphy shows one bioactive zone abou, 18 to 20 cm from origin. Ninhydrin spray reveals two spots ~10 cm and 18 - 20 cm from origin). Racemomycin A sulfate, H. Taniyama and F. Miyoshi, Chem. Pharm. Bull. (Tokyo), 10, 156 (1962), a known streptothricin-like antibiotic used as a standard,shows a wide zone of inhibition 21 - 26 cm. from origin when tested under the same 0 conditions and a duplicate chromatogram sprayed with ninhydride reveals a spot at 24 cm.
d) Paper chromatography of the sulfate salt of component 484-A in the conventional ascending manner at 24C
for 16 hours on Whatman*No~ I paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20 6:24) gives two main spots as detected by ninhydrin Rf 0.15 - 0.16 and Rf 0.22 - 0.24; Racemomycin A sulfate ,! gives a spot with Rf 0.23 - 0.24 under these conditions.
,~ e) Circular paper chromatography [see, M.l. Horowitz20~ and C.P. Schaffner, Anal. Chem., 30 1616 (1958)],of the sulfate , ~ , : -.
salt of component 484-A using Whatman*No. I paper and a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6 24) gives one zone of antimicrqbial activity at Rf 0.40 -0.42 and two zones with Rf 0.30 - 0.32 and Rf 0.40 - 0.42 as~detected . ~ e ' by ninhydrin spray; racemomycin A sulfate gives a spot with Rf 0.44 -0.46 in this system as detected by ninhydrin spray. - ~
* Trade Na~e - -. , .
~, . .
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, ........ . . . . .
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f) A representative infrared absorption spectrum o~ component 484-A in KBr pellet is reprcd~ced in the accompanyina Figure 2 and shows characteristic absorption bands at 3300, 3080 (sh), 2940 (sh), 1710 (sh), 1660, 1550, 1390, 1350, 1310, 1105, 955, 920 and 850 cm~l.
g) The minimum inhibitory concentration of component 484-A
against various microorganisms is listed in Table 1, see below.
h) Component 484-A exhibits a LD50 (i.P.J ~ice) >1.0 g/kg.
This LD50 readily distinguishes this substance from other known streptothricin-like antibiotics.
i~ AmTno acid analysis of component 484-A indicated the presence of a streptolidine moiety and a L-~-lysine moiety in about a I to I ra~ion.
In protection studies mice were infected with ATCC 10536. At one hour and again at five hours a~ter infection, mice were given ~5 mg/kg, (sOc) of component 484-A. The result was complete protection of the mice. ~ -Furthermore, like the complex, component 484-A gives - positive reactions with ninhydrin and siIver nitrate, and negative reactions wi1h Elson-Morgan and Sakaguchi reagents.
The specific rotation ([~]D5) of component 484-A is -25 + 3~
(H20)~ i.e. the specific rotation of several batches range from -22 to -28 (H20). One sample of component 484-A had the specific rotation [~]~5 = -27C (HzO~.
The specific rotation ([a]D25) of several batches of component 484-A ranges from -22 to -28 ~H20). One sample of component 484-A had the specific rotation [a]D = -27C (H20).
Characterizatlon of Antibiotic Component 484-B
Componenf 484-B is characterized by its mode of preparation and the following additional characterization:
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a) Component 484-B; like the complex 484 and the component 484-A, is soluble in water and methanol and substantially insoluble in acetone, ether, benzene, ethyl acetate and chloroform.
b) Component 484-B is convertible to acid addition salts, for example, the inorganic acid addition salts, hydrochlorTde, hydro- ' bromide, sulfate and phosphate, and the organic acid addition salts, fo'r' example`~' a'cetate, pyruvate, tartrate, citrate, malate and aspartate. The salts are readily obtained by treatment of the componenJ with the corresponding organic or inorganic acid.
The salts possess the same antibacterial activity as the component itself and are used in the same manner. -~
c) Paper chromatography of the component 484-B in the conventional descending manner at 24C for 48 hours on Whatman -No. I paper using a solvent system consisting of n-propanol, ' -' ' pyridine, acetic acid and water (6:4:1.5:4) gives a main area of antimicrobial activity, as determined by bioautography on ~gar plates seeded with ~ ~ at 17 cm and minor zones at 24 and 8 cm from the origin.
:,,j . :
d) Circuiar chromatography utilizing Whatman*No. l paper and~ a solv0nt system consisting of n-propanol, pyridine, acetic acid and water (30:20:6~24) gives three zones ' ;~ as detected by ninhydrin (Rf 0.16, 0.29'and 0.42).
e) A representative infrared absorption spectrum of component ' 4B4-B is reproduced in accompanying Figure 3 and shows characteristic 1 bands at 3280, 3070, 2930, 1710, 1655, 1550, 1395, 1075 and 920 crn 1.
f) The minimum inhibitory concentration of component 484-B
` against various microorganisms is listed in Table 1, see below.
.
g) Component 484-B exhibits a LD50 (i P ~ mice) of about 100 mg/kg. - -* Trade Name ,, . ~ , - :
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h) Amino acid an~lysis of component 484-B indicates the presence of a streptolidi.ne moiety and a L-~-lysine rnoiety in about a I to 2 ratio.
The specific rotation ([a~2D5) of this compo~nent is -12 + 2 (H20), i.e. the specific rotation of several batches of component 484-A ranged from -10 to -14 (H20). One sampie of component 484-B had a specific rotation, ~a~D4 = -12 (H20).
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TABLE I
Minimum Inhibitory Concentration (MIC) in mcq/ml for the Antibiotic Substances of this Invention _ _ ~est;MicroorqanismComplex 484 Component 484-A Component 484-B
Staphylococcus pyogenes(penicillin- 0-4 0.8 ~D.I
sens i t i ve ) , Staphylococcus , 'rYes`istant~P i 3.2 0.4 : -Streptococcus -faecalls 50.0 50.0 12.5 - Escherichia coli 3.2 3.2 0.8 Aerobacter aerogenes I o6 1.6 0.4.
, _ , _ . . .
Salmonalla pullorum 50.0 . 50-0 6.4 .
Pseudomonas aeruginosa 50~,0 50.0 12.5 , Proteus mirabilis3.2 3.2 0.8 Proteus vulgaris 1.6 3.2 0.4 : Klebsiella pneumoniae 1.6 1.6 0.4 Serratia ~,arcescens 3.2 3.2 0.8 ' ~, The following examples illustrate ~urther this invention.
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EXhMPlE I
_reparation and Purificatlon of ~ x 48 -M roorqanism StreDtomvces sp. NRRL 5490 was grown and maintained on oatmeal-tomato paste agar slants (T.G. Pridham, et alO, Antibiotic Annual 1956 - 1957, Medical Encyclopedia Inc., ; New York, p. 947) and in Roux bottles containjng the same medium. Good growth was obtained after 7 days of incubation at 28C. Spores from one Roux bottle were washed off and suspended into 50 ml of sterile distilled water. This suspension was used to inoculate the first stage inoculum.
Flrst Staae Inoculum The first stage inoculum medium is an aqueous medium having the following constituents glucose (Cerelose)* 2.0%
` ~ peptone (Neopeptone)* 0.5%
tomato paste 1.0%
corn meal 0.8%
"Blackstrap" molasses 2.0%
sodium chloride 0.3%
pH adjusted to 7.8 withammonium hy~roxide This first stage inoculum medium (3.2 1), contained in a twenty-four litre Florence flask,was sterilized at 121C for I hour, cooied and inoculated with (32 ml, 1% inoculum) of the spore suspension described above. The inoculated flask was incubated for 30 hours at 28C on a reciprocating shaker operating at 65 rev/min t4"-stroke).
Production _t~e ` The production stage was run in 250 liter New Brunswlck fermenters, tModel F-250 made by the New Brunswick Scientific Co., New Brunswick, N.~.~, e~uipped with automatic antifoam addition system and pH recorder-controller. The fermenters were charged ~i - 2:l , *Trade Mark ' ~' '' ' ' ', :. ' .' -'. ' ' :
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t~ P- f 0~ C I
with 160 liters of the same medium as described abové, and lard oil (0.1% v/v, Larex*No. i, Swift Canadian Co., Toronto)~/as added as an antifoam agent before sterilization. The fermenters were sterilized at 121C for 35 minutes. The sterilized and cooled fermenters were inoculated with one flask (3.2 1, 2% inoculum) of first stage inoculum. Incubation temperature 28C;
aeration: 0.5 vol/vol/min; agitation: 250 rev/min. The antifoam agent, used on demand, was Mazur*DF-143PX (Mazur Chemical Corp., Gurnee, Illinois). During the course of fermentation pH was kept at or above 6.0 by automatic addition of 25% sod1um hydroxide solution~
After six days incubation the pH of the fermentation broth reached 7.5 and a 1.3 cm diameter fiIter paper disc in broth gave an inhibition zone of 23 mm on an agar plate seeded :
with a strain of Pseudomonas aeruainosa. At this time the ; packed cell volume (PCV) was 24%. The fermentation was stopped, the pH of the broth was adjusted to 3.0 with sulfuric acid, and ,~ .
diatomaceous earth ~5% v/v) was added. The mixture was subjected to filtration. The filtrate containing the antibiotic principle was retained and the mycelium discarded.
Extraction and Recovery The pH of the fiItrate (volume = 150 liters) was adjusted to 6.5 with I N ammonium hydroxide solution. The filtrate was passed through a coiumn containing 7 liters of Amberlite*lRC 50 NH
resin at a rate of 20 liters per hour. The product was absorbed on the resin and the eluate was rejected. The resin was washed with deionized water until the washings were colorless. The product *Trade Mark ,,~,~j;.~
AHP- ~Jr,~-l-CI
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was eluted from the resin with 12 liters of I N ammonium hydroxide solution. The ammoniurn hydroxide eluate was evaporated to dryness under reduced pressure at 40C. The residue was dissolved in 3 liters of methanol with vigorous stirring. The solution was filtered. The fi~trate containing the active principle was de-colorized by stirr7ng for 20 min. witn 1~ charcoal (Darc~ G-60) and filtered. The char,coal was washed with I liter of methanol with stirring. The methanol fiItrate and washings were evaporated to 500 ml under reduced pressure. Addition of seven volumes of acetone to the methanol concentrate precipitated the antibiotic complex.
Further purification was obtained by dissolving the ~ -latter product (40 9) in 400 ml~ of methanol and slowiy adding 2800 ml of acetone to the vigorously stirred solution. The resulting precipitate was collected. R2petition of tnis , 15 purification step gave the purifTed complex.
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6~;6 Se~ _ation of the Complex 4~4 into Com~onents 484-A and 484-B.
CM-Sephadex*C-25 in the N~4+ form was equilibrated with 0.05 N ammonium acetate (pH 7). The complex (10.0 9), obtained as described in Example 1, was dissoived in a small volume of water and the pH adjusted to 7 with aceti¢ acid. This solution was applied to a column containing 300 ml of the above ion exchanger, CM-Sephade~C-25. The column was eluted stepwise with buffered O.05 M ammonium acetate of increasing pH, the pH being adjusted by the addition of ammonium hydroxide or acetic acid. The eluted fractions were evaporated to dryness under reduced pressure. Accordingly, the column was eluted first with 2 - 3 bed volumes of ammonium acetate buffer (pH 7), followed by 2 - 3 bed volumes of ammonium acetate buffer (pH 9.0). These ' 15 fractions were inactlve and were discarded. The column was eluted next with 3 - 4 bed volumes of ammonium acetate butfer (pH 10). The material obtained had a low order of activity.
Thereafter the column was eluted with 3 - 4 bed volumes of ammonium acetate buffer (pH 10.5). The first I - 1.5 bed volumes of eluate contained material of iow activity and the next I - 2 bed volumes contalned material of high activity. This material was , designated component 484-A.
. ~ , Further elution of the column with the same buffer (3rd and 4th bed volumes), foliowed by elution with 2 - 3 bed volumss of 0.25 N ammonium hydroxide gave material of high activity. ~his material was designated component 484-B .
Component 484-A or 484-B were purified further by dissolving each component in a small volume of 50% methanol-water and passing the solution through a column ofAmberlite*lR~45 (OH-). The .... .
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~r~34~i~66 resulting precipitate was collected, washed with acetone and dried under reduced pressure to give the purified components 4~4-A and 484-B, respectively.
Elementary analysis of component 484-A indicates an empirical formula containing from I - 3 molecules of water per molecule of free ' ' C18-l9H34_36N7_g7_g x I ~ 3 ~21 The hydrochloride salt of component 484-A contains from I - 3 molecules of water and from I - 3 molecules of hydrochloric acid per molecule of free base, viz., i8-19H34 36N7_87_8 x 1-3 H20 x 1-3 HCI.
Elementary analysis of component 484-B indicates an empirical formula containing from I - 5 molecules of water per molecule of free - .
base, viz-, C23-25H44-46N9-loo8-9 2 '~ .
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~r~34~i~66 resulting precipitate was collected, washed with acetone and dried under reduced pressure to give the purified components 4~4-A and 484-B, respectively.
Elementary analysis of component 484-A indicates an empirical formula containing from I - 3 molecules of water per molecule of free ' ' C18-l9H34_36N7_g7_g x I ~ 3 ~21 The hydrochloride salt of component 484-A contains from I - 3 molecules of water and from I - 3 molecules of hydrochloric acid per molecule of free base, viz., i8-19H34 36N7_87_8 x 1-3 H20 x 1-3 HCI.
Elementary analysis of component 484-B indicates an empirical formula containing from I - 5 molecules of water per molecule of free - .
base, viz-, C23-25H44-46N9-loo8-9 2 '~ .
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Claims (8)
1. A process for the production of the antibiotic complex 484 which comprises cultivating Streptomyces sp. NRRL 5490 in a culture medium containing a source of assimilable carbon and assimilable organic nitrogen and mineral salts until a substantial amount of the antibiotic complex is produced by said organism in said culture medium and isolating said , antibiotic complex from said culture medium.
2. The process as claimed in Claim I wherein the cultivation is carried out at a temperature ranging from 20 to 35°C and for a period from two to nine days.
3. The process as claimed in Claim I wherein the isolation of the antibiotic complex is effected by adjusting the pH of the fermentation mixture to about pH 3 with an acid, separating the mycelium from the fermentation mixture, adjusting the mycelium-free fermentation mixture to pH 6.0 to 7.0 by the addition of a base, and treating the mixture with an absorbent, eluting the absorbent with an alkaline solution or an alkaline buffer, to produce an extract and isolating the antibiotic complex from the extract.
4. The process as claimed in Claim 3 wherein the antibiotic complex is subject further to chromatography to separate said antibiotic complex into component 484-A and component 484-B.
5. The process as claimed in Claim 4 wherein the chromatography is effected on a cross-linked dextran based cation exchanger.
6. Antibiotic complex 484 which (a) is soluble in water and methanol and substantially insoluble in acetone, ether, benzene, ethyl acetate and chloro-form, (b) is convertible to acid addition salts, (c) gives two areas of antimicrobial activity 25 and 11 cm from the origin on descending paper chromatography at 24°C for 48 hours on Whatman*No. 1 paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6:20), (d) does not exhibit any characteristic absorption between 220 and 350 nm in its ultraviolet absorption spectrum, (e) shows characteristic Infrared absorption at 3330, 2925, 2875 (sh), 1710, 1650, 1560 (sh), 1450, 1380, 1170, 1080, 1030, 970, 920 and 820 cm (f) inhibits the growth of Staphylococcus pyogenes (penicillin-sensitive), Staphylococcus pyogenes (penicillin-resistant), Streptococcus faecalis, Escherichia coli, Aero-bacter aerogenes, Salmonella Pullorum, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae and Serratia marcescens, (g) exhibits a LD50 (i.p., mice) of 350 - 450 mg/kg and a LD50 (p.o., mice) of >1.0 g/kg, (h) has a specific rotation, [.alpha.]?5 = -25 + 3° (methanol), and * Trade mark (i) gives a positive reaction to ninhydrin, a yellow color with Elson-Morgan reagent (i.e. a negative reaction), a negative reaction with Sakaguchi reagent and a positive reaction with silver nitrate reagent when prepared by the process of Claims 1,2 or 3, or an obvious chemical equivalent thereof.
7. Antibiotic component 484-A which (a) is soluble in water and methanol and substantially insoluble in acetons, ether, benzene, ethyl acetate and chloroform (b) is convertible to acid addition salts, (c) gives one main zone of antimicrobial activity at 23 - 25 cm from origin on descending paper chromatography at 24°C for 48 hours on Whatman*No. 1 paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6:20) and two main spots 13 - 15 cm and 23 - 25 cm from origin in the same system as revealed by ninhydrin spray, (d) is convertible to a sulfate salt giving two main spots, Rf 0.15 - 0.16 and Rf 0.22 - 0.24 on ascending paper chromatography at 24°C for 16 hours on Whatman*No. 1 paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6:24) as detected by ninhydrin, ? (e) is convertible to a sulfate salt giving two zones,as detected by ninhydrin spray, Rf 0.30 - 0.32 and Rf 0.40 -0.42 on circular paper chromatography using Whatman*No. 1 paper and a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6:24) and in the same system gives one zone of antimicrobial activity at Rf 0.40 - 0.42, (f) shows characteristic infrared absorption bands in KBr pellet at 3300, 3080 (sh), 2940 (sh), 1710 (sh), 1660, 1550, 1390, 1310, 1105, 955, 920, and 850 cm-1, *Trade Mark (g) inhibits growth of Staphylococcus pyrogenes (penicillin-sensitive), Staphylococcus pyogenes (penicillin-resistant), Streptococcus faecalis, Escherichia coli, Aerobacter aerogenes, Salmonella pullorum, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae and Serratia marcescens, (h) exhibits a LD50 (i.p., mice) of 1.0 g/kg, (i) has about a 1 to 1 ratio of streptolidine and L-.beta.-lysine moieties as indicated by amino acid analysis, (j) has a specific rotation, [.alpha.]?5 = -25 + 3° (H20), and (k) gives positive reaction with ninhydrin and silver nitrate and negative reactions with Elson-Morgan and Sakaguchi reagents, when prepared by the process of Claims 4 or 5, or an obvious chemical equivalent thereof.
8. Antibiotic component 484-B which (a) is soluble in water and methanol and substantially insoluble in acetone, ether, benzene, ethyl acetate and chloro-form, (b) is convertible to acid addition salts, (c) gives a main area of antimicrobial activity at 17 cm and minor areas at 24 and 8 cm from the origin on descending paper chromatography at 24°C for 48 hours on Whatman*No. 1 paper using a solvent system consisting of n-propanol, pyridine, acetic acid and water (6:4:1.5:4), (d) gives three zones, as detected by ninhydrin spray, Rf 0.16, 0.29 and 0.42, on circular paper chromatography using Whatman*No. 1 paper and a solvent system consisting of n-propanol, pyridine, acetic acid and water (30:20:6:24), *Trade Mark (e) shows characteristic infrared absorption bands in KBr pellet at 3280, 3070, 2930, 1710, 1655, 1550, 1395, 1075 and 920 cm-1, (f) inhibits the growth of Staphylococcus pyogenes (penicillin-sensitive), Staphylococcus pyogenes (penicillin-resistant), Streptococcus faecalis, Escherichia coli, Aerobacter aerogenes, Salmonella pullorum, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Klebsiella Pneumoniae and Serratia marcescens, (g) exhibits a LD50 (i.p., mice) of about 100 mg/kg, (h) has about a 1 to 2 ratio of streptolidine and L-.beta.-lysine moieties as indicated by amino acid analysis, and (i) has a specific rotation, [.alpha.]?4 = -12 ? 2° (H2O), when prepared by the process of Claims 4 or 5, or an obvious chemical equiv-alent thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA234,412A CA1046966A (en) | 1975-08-28 | 1975-08-28 | Antibiotic substances and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA234,412A CA1046966A (en) | 1975-08-28 | 1975-08-28 | Antibiotic substances and preparation thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1046966A true CA1046966A (en) | 1979-01-23 |
Family
ID=4103925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA234,412A Expired CA1046966A (en) | 1975-08-28 | 1975-08-28 | Antibiotic substances and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1046966A (en) |
-
1975
- 1975-08-28 CA CA234,412A patent/CA1046966A/en not_active Expired
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