CA1113028A - Polymyxin f - Google Patents
Polymyxin fInfo
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- CA1113028A CA1113028A CA298,056A CA298056A CA1113028A CA 1113028 A CA1113028 A CA 1113028A CA 298056 A CA298056 A CA 298056A CA 1113028 A CA1113028 A CA 1113028A
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Abstract
ABSTRACT A mixture of antibiotic substances designated polymyxin F is obtained by the cultivation under controlled conditions of a strain of Bacillus circulans, and is active against gram-negative and gram-positive bacteria. This strain of Bacillus circulans has been deposited in the American Type Culture Collection as A.T.C.C. No. 31228.
Description
GC146a A mixture of novel antibiotic substances of unknown chemical structure, designated polymyxin F, is obtained by cultivating a strain of the microorganism Bacillus circulans which has been deposited in the American Type Culture Collection as A.T.C.C. No. 31228. Polymyxin F is a mixture of three acyldecapeptides, said acyldecapeptides differing only in the acyl residue. The mixture of antibiotic substances is active against gram-negative and gram-positive bacteria.
Figure 1 shows the infrared spectrum of the hydro-chloride salt of polymyxin F in potassium bromide.
The microorganism used for the production of poly-myxin F is a strain of Bacillus circulans isolated from the soil. A subculture of the organism may be obtained from the permanent collection of the American Type Culture Collection, Rockville, Maryland. Its accession number in this repository is A.T.C.C. No. 31228.
The characteristics of Bacillùs circulans A.T.C.C.
No. 3-1228 are:
Microscopic: Spore forming bacillus that is gram variable to gram negative. Spores are central to sub-central.
The sporangium is s~ollen. The vegetative rods are not in long chains and are motile. Rods are 0.5-0.7~ x 2.0-5.0~ in size.
GC146a PZ~
Macroscopic: In nutrient broth growth is faintly turbid and confined to the bottom of the tube. No pellicle is ~ormed. No growth occurs above sn oc ~ On nutrient agar, after 3 days of incubation at 28C, growth is thin and adherent to the agar. Occasionally rough and smooth variant colony types are seen within the same culture.
Physiological Characteristics: Catalase is produced.
The Voges-Proskauer test for production of acetylmethylcarbinol is negative. The pH in Voges-Proskauer broth at 7 days is 4.5.
Growth is positive on BBL anaerohic agar made without glucose or Eh indicator. ~cid is formed from glucose, xylose, arabinose and mannitol, but no gas is produced up to 30 days. Crystalline dextrins are not produced. The test for production o~ dihydroxy-acetone from glycerol is negative. Cas~!in is not decomposed.
The above characteristics conf'orm with those of Bacillus circulans, as cited in the monograph of the genus hy Gordon, Haynes and Pang (1973), Agr. Handbook No. ~27 "The Genus sacillus", Agr. Res. Service, ~.S.D.A.
Bacillus circulans A.T.C.C. No. 31228 produces the . . _ .
antibiotic mixture polymyxin F which possesses activity against gram-negative and gram-positive bacteria. To form the anti-biotic mixture polymyxin F according to the preferred method, Bacillus circulans A.T.C.C. No. 31228 is grown at, or near, room temperature (25C) under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable carbo-hydra~e and nitrogen source. The fermentation is carried out until substantial antibiotic activity is imparted to the medium, usually about 60 to 120 hours, preferably about 9n hours.
GC146a ~ fter the fermentation is completed, the beer is acidified, preferably to about pH 2, with an acid such as concentrated hydrochloric acid. Filter aid is added to the acidified fermentation beer, and the whole suspension is filtered. The solids are liberally washed with water, and the washings are then pooled with the filtrate. The washed solids are discarded. The filtrate plus added washings are extracted with water-saturated n-butanol, the butanol extract is concentrated ln vacuo at a temperature below 45C to a small volume, and the concentrate is dissolved in a small volume of methanol. Polymyxin F is precipitated by the addition of acetone, and then washed with acetone and dried 1n vacuo. The precipitate is dissolved in water, absorbed on a weak acid ion-exchange resin, e.~., Amberlite I~C-50 resin (Na ), at pH 6.0 to 7.5, and the antibiotic is eluted from the resin by suspending the resin in methanol-water (1:1) and adjusting the pH to about 1.0 with hydrochloric acid. Elution with methanol-water (1:1) adjusted to about pH 1.5 with concentrated hydrochloric acid is continued until all of the polymyxin F is removed from the resin. The combined eluate is concentrated ln vacuo until the methanol is removed, the concentrate i5 adjusted to pH 10.5 with sodium hydroxide and polymyxin F is extracted into hutanol. The butanol extract is washed with lN hydrochloric acid and concentxated ln vacuo.
The residue is dissolved in methanol and polymyxin F is pre-cipitated with ethyl acetate. The precipitate is washed with ethyl acetate and dried ln vacuo, yielding partially purified polymyxin F. The partially purified antibiotic is dissolved in a mixture of water and methanol, ahsorbed on a column of carboxymethyl cellulose (Na ), and eluted from the column with ~ GC146a a sodium chloride gradient. Active fractions from the major antibiotic component are combined, acidified with l_ hydro-c~loric acid, and extracted with butanol. The butanol extract is concentrated in vacuo and polymyxin F hydrochloride is isolated by precipitation from methanol with ethyl acetate as descrihed above.
Polymyxin F is a mixture of basic antihiotic substances that forms salts with various inorganic and organic acids. The hydrochloride, prepared by the above procedure, can be conven-iently converted to any desired water-soluble salt using anion-exchange resins. Alternatively, the free base can be prepared by extracting a butanol solution of the hydrochloride with dilute aqueous sodium hydroxide and then removing the butanol in vacuo. Salts can be obtained from the residue by neutralization with the appropriate acid.
~ cid hydrolysis o polymyxin F yields a mixture of
Figure 1 shows the infrared spectrum of the hydro-chloride salt of polymyxin F in potassium bromide.
The microorganism used for the production of poly-myxin F is a strain of Bacillus circulans isolated from the soil. A subculture of the organism may be obtained from the permanent collection of the American Type Culture Collection, Rockville, Maryland. Its accession number in this repository is A.T.C.C. No. 31228.
The characteristics of Bacillùs circulans A.T.C.C.
No. 3-1228 are:
Microscopic: Spore forming bacillus that is gram variable to gram negative. Spores are central to sub-central.
The sporangium is s~ollen. The vegetative rods are not in long chains and are motile. Rods are 0.5-0.7~ x 2.0-5.0~ in size.
GC146a PZ~
Macroscopic: In nutrient broth growth is faintly turbid and confined to the bottom of the tube. No pellicle is ~ormed. No growth occurs above sn oc ~ On nutrient agar, after 3 days of incubation at 28C, growth is thin and adherent to the agar. Occasionally rough and smooth variant colony types are seen within the same culture.
Physiological Characteristics: Catalase is produced.
The Voges-Proskauer test for production of acetylmethylcarbinol is negative. The pH in Voges-Proskauer broth at 7 days is 4.5.
Growth is positive on BBL anaerohic agar made without glucose or Eh indicator. ~cid is formed from glucose, xylose, arabinose and mannitol, but no gas is produced up to 30 days. Crystalline dextrins are not produced. The test for production o~ dihydroxy-acetone from glycerol is negative. Cas~!in is not decomposed.
The above characteristics conf'orm with those of Bacillus circulans, as cited in the monograph of the genus hy Gordon, Haynes and Pang (1973), Agr. Handbook No. ~27 "The Genus sacillus", Agr. Res. Service, ~.S.D.A.
Bacillus circulans A.T.C.C. No. 31228 produces the . . _ .
antibiotic mixture polymyxin F which possesses activity against gram-negative and gram-positive bacteria. To form the anti-biotic mixture polymyxin F according to the preferred method, Bacillus circulans A.T.C.C. No. 31228 is grown at, or near, room temperature (25C) under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable carbo-hydra~e and nitrogen source. The fermentation is carried out until substantial antibiotic activity is imparted to the medium, usually about 60 to 120 hours, preferably about 9n hours.
GC146a ~ fter the fermentation is completed, the beer is acidified, preferably to about pH 2, with an acid such as concentrated hydrochloric acid. Filter aid is added to the acidified fermentation beer, and the whole suspension is filtered. The solids are liberally washed with water, and the washings are then pooled with the filtrate. The washed solids are discarded. The filtrate plus added washings are extracted with water-saturated n-butanol, the butanol extract is concentrated ln vacuo at a temperature below 45C to a small volume, and the concentrate is dissolved in a small volume of methanol. Polymyxin F is precipitated by the addition of acetone, and then washed with acetone and dried 1n vacuo. The precipitate is dissolved in water, absorbed on a weak acid ion-exchange resin, e.~., Amberlite I~C-50 resin (Na ), at pH 6.0 to 7.5, and the antibiotic is eluted from the resin by suspending the resin in methanol-water (1:1) and adjusting the pH to about 1.0 with hydrochloric acid. Elution with methanol-water (1:1) adjusted to about pH 1.5 with concentrated hydrochloric acid is continued until all of the polymyxin F is removed from the resin. The combined eluate is concentrated ln vacuo until the methanol is removed, the concentrate i5 adjusted to pH 10.5 with sodium hydroxide and polymyxin F is extracted into hutanol. The butanol extract is washed with lN hydrochloric acid and concentxated ln vacuo.
The residue is dissolved in methanol and polymyxin F is pre-cipitated with ethyl acetate. The precipitate is washed with ethyl acetate and dried ln vacuo, yielding partially purified polymyxin F. The partially purified antibiotic is dissolved in a mixture of water and methanol, ahsorbed on a column of carboxymethyl cellulose (Na ), and eluted from the column with ~ GC146a a sodium chloride gradient. Active fractions from the major antibiotic component are combined, acidified with l_ hydro-c~loric acid, and extracted with butanol. The butanol extract is concentrated in vacuo and polymyxin F hydrochloride is isolated by precipitation from methanol with ethyl acetate as descrihed above.
Polymyxin F is a mixture of basic antihiotic substances that forms salts with various inorganic and organic acids. The hydrochloride, prepared by the above procedure, can be conven-iently converted to any desired water-soluble salt using anion-exchange resins. Alternatively, the free base can be prepared by extracting a butanol solution of the hydrochloride with dilute aqueous sodium hydroxide and then removing the butanol in vacuo. Salts can be obtained from the residue by neutralization with the appropriate acid.
~ cid hydrolysis o polymyxin F yields a mixture of
2,4-diaminobutyric acid (Dab), threonine (Thr), serine (Ser), isoleucine (Ile), leucine (Leu) and fatty acid in an approx-imate molar ratio of 5~ 1;2:1. The fatty acid is a mixture of 6-methyloctanoic acid, isooctanoic acid and octanoic acid.
Polymyxin F is thus a mixture of three acyldecapeptides, the components of which differ in the acyl residue. The compo-sitions and empirical formulae of these components are as follows:
Polvmyxin FlPolymyxin F2Polymvx _ F3 Dab(5) Dab(5) Dab(5) Thr(l) Thr(l) Thr(l) Ser(l) Ser(l) Ser(l) Ile(l) Ile(l) Ile(l) Leu(2) Leu(2) Leu(2) 6-Methyloctanoic acid (1) Isooctanolc acid (1) Octanoic acid (1) C54 lOlN1513 53 99 1513 C53~l99~15O13 GC146a a.3~
Polymyxin Fl is the most abundant component and poly-myxin F3 the least abundant component. Each component has four primary amino groups (the y-amino groups of four of the Dab residues) and no other ionic functionality. Polymyxin F forms acid salts having stoichiometry consistent with this chemical make-up.
The following example further illustrates the prepara-tion of polymyxin F.
GCl46a Example l Yeast beef agar slants are seeded with Bacilllls circulans A.T.C.C. No. 31228, incubated overnight at 30C
and used to inoculate lOO ml of an aqueous soybean meal medium contained in 500 ml Erlenmeyer flasks. The composition of the germination medium is:
Medium Grams Glucose 50-0 *Nutrisoy Flour 15.Q
Soluble Starch 15.0 CaC03 10. 0 COCl2 6H2 S
Distilled water to l liter The medium is sterilized for 30 minutes at 121C and at 15 lbs steam pressure prior to use. The inoculated germina~ion flasks are incubated at 25C for 72 hours on a rotary shaker, oper-ating at 280 r.p.m. with a 2-inch throw.
A 5% (v/v) transfer is made from the germination flasks to lO liters of medium contained in a 14 liter glass vessel with the medium and operating conditions described below:
Medium Grams A. *Nutrisoy Flour 150 Soluble Starch 150 CaCO3 lO0 CC12- 6H2 O. 05 Distilled water to 9 liters B. Glucose, 50% in water l liter The ingredients in A are sterilized separately from the glucose of B. Both are sterilized at 15 lbs. pressure at 121C Eor 15 minutes prior to use. The inoc~llum, 500 ml, is added to * Trade Mark ,~ .
-~ C7Cl46a 10 liters of medium and incubated 90 hours. During incubation, the broth is aerated at the rate o~ 1.4 volumes of air per volume of broth per minute and stirred at 750 rpm.
The fermentation broth (10 liters~ is adjusted to pH
2O0 with concentrated hydrochloric acid (75 ml). The solids are separated by centrifugation at 9,000 rpm. and washed with two l~ er portions of water. The washings are combined with the supernate to give 10 liters. The washed cake (750 g) is discarded.
The supernate (10 liters) is extracted three times with
Polymyxin F is thus a mixture of three acyldecapeptides, the components of which differ in the acyl residue. The compo-sitions and empirical formulae of these components are as follows:
Polvmyxin FlPolymyxin F2Polymvx _ F3 Dab(5) Dab(5) Dab(5) Thr(l) Thr(l) Thr(l) Ser(l) Ser(l) Ser(l) Ile(l) Ile(l) Ile(l) Leu(2) Leu(2) Leu(2) 6-Methyloctanoic acid (1) Isooctanolc acid (1) Octanoic acid (1) C54 lOlN1513 53 99 1513 C53~l99~15O13 GC146a a.3~
Polymyxin Fl is the most abundant component and poly-myxin F3 the least abundant component. Each component has four primary amino groups (the y-amino groups of four of the Dab residues) and no other ionic functionality. Polymyxin F forms acid salts having stoichiometry consistent with this chemical make-up.
The following example further illustrates the prepara-tion of polymyxin F.
GCl46a Example l Yeast beef agar slants are seeded with Bacilllls circulans A.T.C.C. No. 31228, incubated overnight at 30C
and used to inoculate lOO ml of an aqueous soybean meal medium contained in 500 ml Erlenmeyer flasks. The composition of the germination medium is:
Medium Grams Glucose 50-0 *Nutrisoy Flour 15.Q
Soluble Starch 15.0 CaC03 10. 0 COCl2 6H2 S
Distilled water to l liter The medium is sterilized for 30 minutes at 121C and at 15 lbs steam pressure prior to use. The inoculated germina~ion flasks are incubated at 25C for 72 hours on a rotary shaker, oper-ating at 280 r.p.m. with a 2-inch throw.
A 5% (v/v) transfer is made from the germination flasks to lO liters of medium contained in a 14 liter glass vessel with the medium and operating conditions described below:
Medium Grams A. *Nutrisoy Flour 150 Soluble Starch 150 CaCO3 lO0 CC12- 6H2 O. 05 Distilled water to 9 liters B. Glucose, 50% in water l liter The ingredients in A are sterilized separately from the glucose of B. Both are sterilized at 15 lbs. pressure at 121C Eor 15 minutes prior to use. The inoc~llum, 500 ml, is added to * Trade Mark ,~ .
-~ C7Cl46a 10 liters of medium and incubated 90 hours. During incubation, the broth is aerated at the rate o~ 1.4 volumes of air per volume of broth per minute and stirred at 750 rpm.
The fermentation broth (10 liters~ is adjusted to pH
2O0 with concentrated hydrochloric acid (75 ml). The solids are separated by centrifugation at 9,000 rpm. and washed with two l~ er portions of water. The washings are combined with the supernate to give 10 liters. The washed cake (750 g) is discarded.
The supernate (10 liters) is extracted three times with
3-liter portions of water-saturated n-butanol. The comhined butanol extract (7 liters) is concentrated ln vacuo, at a temperature less than 45C., to a small volume (lOn ml).
The butanol-extract concentrate Erom two 10-:Liter ferment-ations, obtained as described above, is diluted with methanol (300 ml) and added to 7.5 liters of acetone. The resulting precipitate is separated, washed with acetone, and dried ln vacuo, giving 13.6 g of solid.
The acetone-insoluble powder, 13D 6 g, is dissolved in 300 ml of water and stirred with 135 g of Amberlite IRC-50 ion-exchange resin (H~ form) for 12 hours, adding 5 N sodium hydro~ide as necessary to maintain the p~ between 6.0 and 7.5.
The resin is separated, washed with methanol-water (1:1) and ~hen stirred for 2 hr with 400 ml of methanol-water (1:1~
maintaining the pH at 1.0 by the addition of concentrated hydro-chloric acid. The slurry is then placed in a column and the resin eluted with methanol-water (1:1) adjusted to pll 1.5 wlth concentrated hydrochloric acid until the effluent from the column has ne~ligible antibiotic activity. The combined eluate (ca.
600 ml) is concentratecl in vacuo to remove the methanol, yielding GC146a an aqueous solution of polym~xin F.
An aqueous solution of polymyxin F is mixed with butanol and adjusted to pH 10.5 with 5N sodium hydroxide. The butanol phase is separated and washed twice with 0.01 N sodium hydroxide and then three times with lN hydrochloric acid, back-extracting the acid washes with butanol. The combined butanol extract is concentra~ed in vacuo. The residue is dissolved in methanol and added to ethyl acetate. The resulting precipitate is washed with ethyl acetate and dried in vacuo givin~ 0.94 g o~ polymyxin F, which is dissolved in 35 ml of methanol-water, 5:2, and applied to a 2.5 X 50 cm column of Whatman CM52 carboxymethyl cellulose in the sodium form. The column is eluted at a rate of 3 ml/minute, first with 120 ml of water and then with a linear gradient prepared from 4 liters of 0.15N sodium chloride and 4 liters of 0.30N
sodium chloride. Fractions are collected and assayed by paper-disc agar diffusion assay to locate the main antihiotic peak.
The fractions comprising the main peak of activity are combined.
The resultin~ solution is acidified with hydrochloric acid and washed with chloroform. The antibiotic is then extracted from the aqueous phase with butanol. The butanol is removed ln vacuo and the residue converted to a powder, 0.44 g, by precipitation from me~hanol with ethyl acetate as descrihed ahove.
Analysis: Calcd- for C54H101~1513 H, 8.05; N, 15.99; Cl, ln.79 Found: C, 49.68; H, 8.05; N, 16.13;
Cl, 10.96 The infrared spectrum o~ polymyxin F as the hydrochloride
The butanol-extract concentrate Erom two 10-:Liter ferment-ations, obtained as described above, is diluted with methanol (300 ml) and added to 7.5 liters of acetone. The resulting precipitate is separated, washed with acetone, and dried ln vacuo, giving 13.6 g of solid.
The acetone-insoluble powder, 13D 6 g, is dissolved in 300 ml of water and stirred with 135 g of Amberlite IRC-50 ion-exchange resin (H~ form) for 12 hours, adding 5 N sodium hydro~ide as necessary to maintain the p~ between 6.0 and 7.5.
The resin is separated, washed with methanol-water (1:1) and ~hen stirred for 2 hr with 400 ml of methanol-water (1:1~
maintaining the pH at 1.0 by the addition of concentrated hydro-chloric acid. The slurry is then placed in a column and the resin eluted with methanol-water (1:1) adjusted to pll 1.5 wlth concentrated hydrochloric acid until the effluent from the column has ne~ligible antibiotic activity. The combined eluate (ca.
600 ml) is concentratecl in vacuo to remove the methanol, yielding GC146a an aqueous solution of polym~xin F.
An aqueous solution of polymyxin F is mixed with butanol and adjusted to pH 10.5 with 5N sodium hydroxide. The butanol phase is separated and washed twice with 0.01 N sodium hydroxide and then three times with lN hydrochloric acid, back-extracting the acid washes with butanol. The combined butanol extract is concentra~ed in vacuo. The residue is dissolved in methanol and added to ethyl acetate. The resulting precipitate is washed with ethyl acetate and dried in vacuo givin~ 0.94 g o~ polymyxin F, which is dissolved in 35 ml of methanol-water, 5:2, and applied to a 2.5 X 50 cm column of Whatman CM52 carboxymethyl cellulose in the sodium form. The column is eluted at a rate of 3 ml/minute, first with 120 ml of water and then with a linear gradient prepared from 4 liters of 0.15N sodium chloride and 4 liters of 0.30N
sodium chloride. Fractions are collected and assayed by paper-disc agar diffusion assay to locate the main antihiotic peak.
The fractions comprising the main peak of activity are combined.
The resultin~ solution is acidified with hydrochloric acid and washed with chloroform. The antibiotic is then extracted from the aqueous phase with butanol. The butanol is removed ln vacuo and the residue converted to a powder, 0.44 g, by precipitation from me~hanol with ethyl acetate as descrihed ahove.
Analysis: Calcd- for C54H101~1513 H, 8.05; N, 15.99; Cl, ln.79 Found: C, 49.68; H, 8.05; N, 16.13;
Cl, 10.96 The infrared spectrum o~ polymyxin F as the hydrochloride
4) in KBr is shown in Figure 1.
GC146a , . ~
~L~3~
The electrophoretic mobility of polymyxin F on paper, using a buffer consisting of 0.05~ sodium formate in formic acid/t-butanol/water (1:2:7) is 0.84 relative to phloro-glucinol as an electroosmotic indicator (mo~ility 0.0) and polymyxin B (mobility 1.00).
Paper-partition chromatography of polymyxin F on Whatman #1 paper, using the upper phase of butanol/acetic acid/water (4:1:5) ~ives an Rf value of 0.72.
Polymyxin F is soluble in water and methanol and insoluble in acetone, ether, ethyl acetate, benzene and the like.
Hydrolysis of 0.09 mg of polymyxin F in 6N hydrochloric acid at 110C for 16 hours yields a mixture containing 0.326 micromoles of 2,4-diaminobutyric acid, 0.066 micromoles of threonine, 0.063 micromoles of serine, 0.049 micromoles of isoleucine and 0.141 micromoles of leucine as shown by conven-tional Stein-Moore analysis. Gas chromatographic analysis also shows the presence, in descending quantity, o 6-methyl-octanoic acid, isooctanoic acid and octanoic acid.
Specific rotations of polymyxin F hydrochloride (1:4) 20 are as follows:
_ ~ ~] (~ 0.5 in 0 5N HCl) 589 nm -43 The melting point of polymyxin F hydrochloride (1:4), determined in an evacuated capillary, is 213 to 219C.
The UV spectrum of polymyxin F hydrochloride (1:4) in water has no maximum at wavelengths greater than 200 nm;
GC146a there is, however, end absorption with an ElCm at 220 nm of 47.
Biological Activity Two-fold tube dilution assays with several microorganisms show the following results. The polymyxin F used in this study is the hydrochloride (1:4).
Organism MIC_(~g/ml) Staphylococcus aureus FDA 209P 50 Streptococcus EY~ C 203 6 3 10 Escherichia coli ATCC 10536 1.2 -Escherichia coli SC 8294 2.4 Pseudomonas aeruginosa SC 8329 3.1 Organisms from the Squibb Culture Collection A comparison of the activities of polymyxins B and F
show that polymyxin F has substantial activity against some organisms that are resistant to polymyxin B.
MIC (~g/ml) or~anism Polymyxin BPolym~xin F
Escherichia coli SC8599 0.1 1.2 _ 20 Escherichia coli SC8600 a>100 18.7 *
Escherichia coli SC9251 0.06 0.4 , Escherichia coli SC9252 b18.7 9.4 Escherichia coli SC9253 b50.0 12.5 -*
Organisms from the Squihh Culture Collection.
~olymyxin-resistant variant of E. coli SC3599 Polymyxin-resistant variants of E. coli SC9251 Testing with mice that had been infected intraperitoneally with Escherichia coli SC8294 suspended in 5~ hog gastric mucin in an amount 500 times the LD50 shows that 50~ survive after subcutaneous injection of 4.2 mg/kg of polymyxin F hydrochloride (1:4) one hour post infection. None of the mice survive the infection when the antibiotic is not administered.
GC146a , . ~
~L~3~
The electrophoretic mobility of polymyxin F on paper, using a buffer consisting of 0.05~ sodium formate in formic acid/t-butanol/water (1:2:7) is 0.84 relative to phloro-glucinol as an electroosmotic indicator (mo~ility 0.0) and polymyxin B (mobility 1.00).
Paper-partition chromatography of polymyxin F on Whatman #1 paper, using the upper phase of butanol/acetic acid/water (4:1:5) ~ives an Rf value of 0.72.
Polymyxin F is soluble in water and methanol and insoluble in acetone, ether, ethyl acetate, benzene and the like.
Hydrolysis of 0.09 mg of polymyxin F in 6N hydrochloric acid at 110C for 16 hours yields a mixture containing 0.326 micromoles of 2,4-diaminobutyric acid, 0.066 micromoles of threonine, 0.063 micromoles of serine, 0.049 micromoles of isoleucine and 0.141 micromoles of leucine as shown by conven-tional Stein-Moore analysis. Gas chromatographic analysis also shows the presence, in descending quantity, o 6-methyl-octanoic acid, isooctanoic acid and octanoic acid.
Specific rotations of polymyxin F hydrochloride (1:4) 20 are as follows:
_ ~ ~] (~ 0.5 in 0 5N HCl) 589 nm -43 The melting point of polymyxin F hydrochloride (1:4), determined in an evacuated capillary, is 213 to 219C.
The UV spectrum of polymyxin F hydrochloride (1:4) in water has no maximum at wavelengths greater than 200 nm;
GC146a there is, however, end absorption with an ElCm at 220 nm of 47.
Biological Activity Two-fold tube dilution assays with several microorganisms show the following results. The polymyxin F used in this study is the hydrochloride (1:4).
Organism MIC_(~g/ml) Staphylococcus aureus FDA 209P 50 Streptococcus EY~ C 203 6 3 10 Escherichia coli ATCC 10536 1.2 -Escherichia coli SC 8294 2.4 Pseudomonas aeruginosa SC 8329 3.1 Organisms from the Squibb Culture Collection A comparison of the activities of polymyxins B and F
show that polymyxin F has substantial activity against some organisms that are resistant to polymyxin B.
MIC (~g/ml) or~anism Polymyxin BPolym~xin F
Escherichia coli SC8599 0.1 1.2 _ 20 Escherichia coli SC8600 a>100 18.7 *
Escherichia coli SC9251 0.06 0.4 , Escherichia coli SC9252 b18.7 9.4 Escherichia coli SC9253 b50.0 12.5 -*
Organisms from the Squihh Culture Collection.
~olymyxin-resistant variant of E. coli SC3599 Polymyxin-resistant variants of E. coli SC9251 Testing with mice that had been infected intraperitoneally with Escherichia coli SC8294 suspended in 5~ hog gastric mucin in an amount 500 times the LD50 shows that 50~ survive after subcutaneous injection of 4.2 mg/kg of polymyxin F hydrochloride (1:4) one hour post infection. None of the mice survive the infection when the antibiotic is not administered.
Claims (2)
1. A process for the preparation of polymyxin F, or an acid salt thereof, which comprises fermenting Bacillus cir-culans A.T.C.C. No. 31228 under submerged aerobic conditions in an aqueous nutrient medium comprising an assimilable car-bon source and an assimilable nitrogen source, and isolating therefrom polymyxin F, or an acid salt thereof, said poly-myxin F being a mixture of three basic peptides comprising in an approximate molar ratio of 5:1:1:1:2:1 2,4-diaminobu-tyric acid, threonine, serine, isoleucine, leucine, and a mixture of the acyl residues of 6-methyloctanoic acid, iso-octanoic acid and actanoic acid; polymyxin F hydrochloride (1:4) having the infrared spectrum in potassium bromide as shown in Figure 1 and the following physical characteristics:
approximate elemental analysis C, 49.68; H, 8.05; N, 16.13;
Cl, 10.96; and melting point of about 213°C to 219°C, in vacuo.
approximate elemental analysis C, 49.68; H, 8.05; N, 16.13;
Cl, 10.96; and melting point of about 213°C to 219°C, in vacuo.
2. Polymyxin F, or an acid salt thereof, said polymy-xin F being a mixture of three basic peptides comprising in an approximate molar ratio of 5:1:1:1:2:1 2,4-diaminobutyric acid, threonine, serine, isoleucine, leucine, and a mixture of the acyl residues of 6-methyloctanoic acid, isooctanoic acid and octanoic acid; polymyxin F hydrochloride (1:4) hav-ing the infrared spectrum in potassium bromide as shown in Figure 1 and the following physical characteristics: approx-imate elemental analysis C, 49.68; H, 8.05; N, 16.13; Cl, 10.96; and melting point of about 213°C to 219°C, in vacuo, when prepared by the process of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA298,056A CA1113028A (en) | 1978-03-02 | 1978-03-02 | Polymyxin f |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA298,056A CA1113028A (en) | 1978-03-02 | 1978-03-02 | Polymyxin f |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1113028A true CA1113028A (en) | 1981-11-24 |
Family
ID=4110901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA298,056A Expired CA1113028A (en) | 1978-03-02 | 1978-03-02 | Polymyxin f |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1113028A (en) |
-
1978
- 1978-03-02 CA CA298,056A patent/CA1113028A/en not_active Expired
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry | ||
| MKEX | Expiry |
Effective date: 19981124 |