Note: Descriptions are shown in the official language in which they were submitted.
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.
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~ 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
4--
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
,~ .
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~ 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
4) in KBr is shown in Figure 1.
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~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
578 -47
546 -54
436 -97
365 -160
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.
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