Note: Descriptions are shown in the official language in which they were submitted.
1220746
SY-1727
-- 2
BACKGROUND OF ~HE INVENTION
(1) Field of the Invention
This invention relates to a new cyclic depsipeptide
antibiotic and a method for its production and recovery. Also
provided are pharmaceutical compositions containing the new
antibiotic and methods for using said antibiotic (and compo-
sitions thereof) as an antimicrobial and antitumor agent.
(2) Description of the Prior Art
There is disclosed in U.K. Patent Application
2,050,384A an antitumor antibacterial complex designated
BBM-928 and its production by fermentation of a new strain of
actinomycetes designated strain G455-101 (ATCC 31491). This
reference discloses that the BBM-928 complex has at least
six components designated BBM-928 A, B, C, D, E and F.
Components A, B, C and D were fully characterized in the
reference while components E and F are characterized only by
their Rf values in two silica gel TLC systems and by ln vitro
antimicrobial activities against certain aerobic bacteria.
The BBM-928-producing organism strain G455-101 was
later determined to be a new species of the genus Actinomadura
and designated Actinomadura luzonensis nov. sp. (see
_ Antibiotics 33(10): 1098-1102, 1980).
J Am. Chem. Soc. 103: 1241 (1981) discloses the
structures of BBM-928 A, B and C as does Peptide Chemistry
1980, K. Okawa (Ed.), Protein Research Foundation, Osaka, Japan,
pg. 119-124 (1981) and J. Antibiotics 34(2): 148-159 (1981).
The production, isolation, characterization and
antitumor activity of the BBM-928 components are disclosed in
_ Antibiotics 33(10): 1087-1097 (1980). As in U.K. Application
2,050,384A, components E and F are characterized only
~.-
lZZ0746
by their Rf values in two silica gel TLC systems.
A proposed mechanism of the antitumor activityof BBM-928A is disclosed in Biochemistry 19: 5537 (1980).
~ he BBM-928 antibiotics are presently named the
luzopeptin antibiotics after the producing species. As
used herein, therefore, luzopeptin A, B, C, etc. are ide~tical
to BBM-928A, B, C, etc. previously used in the literature.
DESCRIPTION OF THE DRAWINGS
:
FIG. l shows the infrared absorption spectrum of
luzopeptin E2 when pelleted in KBr.
FIG. 2 shows the proton magnetic resonance spectrum
of luzopeptin E2 dissolved in CDC13.
FIG. 3 shows the carbon-13 nuclear magnetic resonance
spectrum of luzopeptin E2 dissolved in CDC13.
SUMMARY OF THE INVENTION
This invention relates to a novel antitumor antibiotic
designated luzopeptin E2 and to a process for its preparation
and isolation in a purified state free of co-produced substances.
The antibiotic is obtained by cultivating a luzopeptin E2-
producing strain of Actinomadura luzonensis, preferably
Actinomadura luzonensis ATCC 31491 or a mutant thereof, in
an aqueous medium containing assimilable sources of carbon
and nitrogen under submerged aerobic conditions until a
substantial amount of luzopeptin E2 is produced by said
organism in said culture medium and recovering the luzopeptin
E2 from the culture medium in a purified form substantially
free of co-produced substances.
Luzopeptin E2 exhibits both antimicrobial and
antitumor activities.
12Z074~
DETAILED DESCRIPTION
Luzopeptin E2 provided by the present invention
has been found to be a minor component of the luzopeptin
(or BBM-928) antibiotic complex disclosed in U.K. Application
2,050,384A. That application discloses the fermentation of
Actinomadura luzonensis sp. nov. to produce a luzopeptin
antibiotic complex and the separation of such complex into
si~ bioactive components now called luzopeptins A, B, C, D,
E and F. There is no disclosure, however, of ~he antibiotic
component luzopeptin E2 which has now been found to be co-
produced in the fermentation of Actinomadura luzonensis.
The new antibiotic of the present invention has
been isolated from the Actinomadura luzonensis fermentation
broth in an essentially pure form and has been characterized
by its physical, chemical and biological properties as
described in more detail below.
Studies carried out by collea~ues of the present
inventors a~ter their isolation and characterization of
luzopeptin E2 have established that luzopeptin E (see, for
example, U.K. Application 2,050,384A) is not in fact a single
antibiotic component but is instead a mixture of components
including luzopeptin E2.
Properties of Luzopeptin E2
Luzopeptin E2 is a cyclic depsipeptide antibiotic
containing a ~uinoline nucleus as the chromophore. From
spectral and chemical analysis, luzopeptin E2 has been
determined to have the structural formula
:i220746
H 3 C CH 3
O-C-~-CH2-CO-N-CB2--CO-N~ L
3 ~ D "l CO
~\N~--Co-N~-cH-co-l ~ o
I H2 B~ ~2
CO Ç-CO-CH-NH-CO~
L CH--N--CO-C~2-7 CO-~H2 ~ H OCH3
3 C~3 O
H3C CH3
~ uzopeptin E2 is a white solid having a melting
point of 201-203~C, a molecular weight of 1315.3732 and a
molecular formula C60H78N14O20. Elemental analysis indicates
the following average percentages by weight:
carbon - 53.19
hydrogen - 5.40
nitrogen - 12.92
oxygen (by difference) - 28.49
The infrared absorption spectrum of luzopeptin E2
when pelleted in KBr is shown in FIG. 1 of the accompanying
drawings. Characteristic infrared absorption bands are
exhibited at the following frequencies expressed in reciprocal
centimeters: 3470, 3370, 2980, 2983, 2850, 1740, 1645, 1520,
1418, 1350, 1285, 1230, 1195, llS0, 1128, 1105, 1065, 1025,
905, 885, 795, 785 and 750.
Proton magnetic resonance and carbon-13 nuclear
magnetic resonance spectra of luzopeptin E2 were determined
with a Bruker Model WM-360 spectrometer operating at 360 MHz
and 90 ~z respectively. The PMR and CMR spectra are shown
as FIG.'s 2 and 3 of the drawings.
In a solvent system consisting of xylene-methyl
ethyl ketone -methanol (5:5:1 v/v), luzopeptin E2 exhibits
an Rf value of 0.33 as determined by silica gel thin layer
chromatography~
-
122074~;
The key structural feature of luzopeptin E2
which distinguishes it from known luzopeptin components
is the presence of a hexahydropyridazine moiety instead
of the tetrahydropyridazine group.
Preparation of Luzopeptin E2
Luzopeptin E2 may be produced by cultivating a
luzopeptin E2-producing strain of Actinomadura luzonensis,
preferably a strain of Actinomadura luzonensis having the
identifying characteristics of ATCC 31491 or a mutant
thereof, under submerged aerobic conditions in an aqueous
nutrient medium.
The producing organisms for luzopeptin E2 and
the fermentation conditions for obtaining it are as disclosed
in U.K. Patent Application 2,050,384A.
While the preferred producing organism is
Actinomadura luzonensis strain G455-101 (ATCC 31491), any
luzopeptin E2-producing strain or mutant of the preferred
organism which can be produced from such organism by conven-
tional means such as x-radiation, ultraviolet radiation,
treatment with nitrogen mustards, phage exposure, and the
like, is intended to be included within the scope of the
present invention.
The producing organism is grown in a conventional
nutrient medium containing an assimilable carbon source such
as starch, glucose, dextrin, maltose, lactose, sucrose,
fructose, mannose, molasses, glycerol and the like. The
nutrient medium should also contain an assimilable nitrogen
source such as protein, protein hydrolysate, polypeptides,
amino acids, corn steep liquor, c:asein, urea and the like as
lZ20746
well as nutrient inorganic salts which provide inorganic
anions and cations such as potassium, sodium, ammonium,
calcium, sulfate, carbonate, phosphate, chlorid-e, nitrate,
and the like.
While not essential for production and recovery
of luzopeptin E2, it is preferred to add a sulfur-containing
compound selected from the group consisting of D,L-methionine,
S-methyl-cysteine, S-ethyl-L-cysteine, D,L-ethionine, D-
methionine, L-methionine, acetyl-methionine, 2-aminoethanethiol,
2-hydroxy-4-(methylthio)butyric acid, D,L-O-methyl-serine,
L-cysteine and L-methionyl glycine to the nutriènt medium.
The preferred sulfur-containing compounds are D,L-methionine
and S-methyl-cysteine. Such sulfur-containing compounds
may be added for example, in a concentration of from about
0.05% to about 0.5% (wt/vol) of the fermentation medium with
the most preferred concentrations for D,L-methionine and S-
methyl-cysteine being 0.15~ and 0.1%, ~espectively. Addition
of the above-mentionéd sulfur-containing compound is found
to increase the production of luzopeptin E2 so that it can
be more readily assayed and isolated.
In producing luzopeptin E2, any temperature
conducive to satisfactory growth of the producing organism
may be employed. Temperatures ranging from about 20 to
45C are operable with a temperature range of 27 to 35~C
most preferred. Maximum production of luzopeptin E2 is
generally obtained after about 72 to 96 hours.
Conventional methods are employed in the fermentation
process. For example, preparation of small amounts is
conveniently carried out in shake flasks or by surface cultures.
Preparation of large amounts is preferably carried out under
submerged aerobic conditions in sterile tanks. With tank
fermentation a vegetative inoculum is first produced in a
nutrient broth by inoculating the broth culture with a spore
from the organism to provide a young active seed culture which
is then aseptically transferred to the fermentation tank
medium. Aeration in tanks and bottles may be provided by
_
~220746
-- 8 --
forcing sterile air through or onto the surface of the
fermenting medium with further agitation in tanks provided
by a mechanical impeller. Anti-foaming agen~s such as
silicone oil, soybean oil and lard oil may be added as needed.
The course of the fermentation can be followed
by assaying the fermentation medium from time to time
against an organism sensitive to luzopeptin E2 such as
Bacillus subtilis ATCC 6633. Luzopeptin E2 may also be
detected by W observation at 360 nm or by its Rf value of
0.37 in the silica gel TLC system xylene-methyl ethyl ketone-
methanol t9:9:1 v/v).
- Isolation of Luzopeptin E2
Luzopeptin E2 may be isolated from the fermentation
broth by conventional procedures such as solvent extraction
procedures and chromatographic procedures as more fully
described in Example 1. Such procedures allow the luzopeptin
E2 component to be recovered in a purified form substantially
free of co-produced products.
Biological Activity Data
The _ vitro minimum inhibitory concentrations
(MIC) of luzopeptin E2 were determined by the serial agar
dilution method. Mueller-Hinton agar was generally used
for gram-positive and gram-negative bacteria except fGr
acid-fast bacteria in which case No. 1001 medium (3% glycerol,
0.3% sodium L-glutamate, 0.2% peptone, 0.31% Na2HPO4, 0.1%
XH2PO4, 0.005% ammonium citrate, 0.001% MgSO4 and 1.5% agar)
was used. The results are shown in Table 1 in comparison with
the activity of luzopeptin A.
~Z~0746
Table 1
In Vitro Antimicrobial Activity of Luzopeptin E2
M in mcg/ml
Test Organism Luzopeptin E2 Luzopeptin A
Staphylococcus aureus 209P 0.4 0.4
Staphylococcus aureus Smith 0.4 0.8
Streptococcus pyogenes S-23 <0.05 0.2
Sarcina lutea PCI 1001 0.1 0.2
_ _ _ .
Micrococcus flavus D12 0.2 0.2
CorYnebacte-r-um xerosis 53K-I 0.B 0.8
Bacillus subtilis PCI 219 0.2 0.8
_
Bacillus megaterium D-2 - -- 0.2 0.2
Bacillus anthracis A9540 0.1 0.2
Escherichia coli NIHJ >100 >100
Klebsiella pneumoniae D-ll >100 >100
Proteus vulgaris A9436>100 >100
Pseudomonas aeruginosa A9930 >100 >100
Mycobacterium sm~gmatis 607 0 8 0 4
Mycobacterium phlei D881.6 0.4
~ZZ0746
-- 10 --
The antitumor activity of luzopeptin E2 against
leukemia P388 was evaluated in BDFl mice by previously
reported procedures (Can. Chem. Rep. _ : 479, 1966 and
Can. Chem. ~. 3: 1, 1972). Results of this test are
shown below in Table 2 along with results for luzopeptin A.
Table 2
Effect of Luzopeptin E2 on P-388
Lymphatic Leukemia
Tumor Inhibition
CompoundDose (mg/kg/day) MST (% T/C)
.
Luzopeptin E2 64 Toxic
32 144
16 167
8 122
4 122
2 100
100
Luzopeptin A64 156
32 161
16 144
8 144
4 128
2 111
Treatment: Once daily for nine injections
Host : BDFl t mice
Evaluation: MST=median survival time
% T/C=MST treated/MST control x 100
Criterion : T/C > 125 considered significant antitumor effect
Toxicity : <4/6 survivors, day 5
~220746
In a second test the antitumor activlty of
luzopeptin E2 was evaluated against leukemia P388 in
BDF mice by the method reported in J. Antibiotlcs 33:
1087-1097 (1980). Luzopeptin A was comparatively tested
as a reference compound. As shown in Table 3, luzopeptins
A and E2 showed nearly equivalent antitumor activity.
The intraperitoneal toxicity determined after multiple
dosing (qd 1~9) of luzopeptin A and E2 is shown in the
last line of Table 3. The toxicity of luzopeptin E2 is
almost 1/2 that of luzopeptin A.
Table 3
Antitumor Actitity of Luzopeptin A
on P388_Leukemia
Dose* (ip) Antitumor activity (% T/C% in MST)
in mg/kg/day Luzopeptin A Luzopeptin E2
O. 1 -- _
0.03 89 143
0.01 174 181
0.003 168 149
0.001 141 129
0.0003 121 117
MED*(mg/kg/day) 0.001 0.001
LD50*(mg/kg/day) 0.019 0.034
* dosing schedule : qd 1~9
12207~6
- 12 -
As indicated by the antimicrobial and mouse
tumor data provided above, luzopeptin E2 is useful as an
antibiotic and also as an antitumor agent for inhibition
of mammalian malignant tumors such as P388 leukemia. The
invention includes within its scope pharmaceutical compositions
containing an effective antimicrobial or tumor-inhibiting
amount of luzopeptin E2 in combination with an inert
pharmaceutically acceptable carrier or diluent. Such
compositions may also contain other active antimicrobial or
antitumor agents and may be made up in any form appropriate
for the desired route of administration. Examples
of such compositions include solid compositions for oral
administration such as tablets, capsules, pills, powdexs
and granules, liquid compositions for oral administration
such as solutions, suspensions, syrups or elixers and
preparations for parenteral administration such as sterile
solutions suspensions or emulsions. They may also be
manufactured in the form of sterile solid compositions which
can be dissolved in sterile water, physiological saline or
some other sterile injectable medium immediately before use.
According to another aspect of the invention, a
method is provided for therapeutically treating a mammalian
host affected by a microbial infection or by a malignant
tumor (e.g. P388 leukemia) which comprises administering
to said host an effective antimicrobial or tumor-inhibiting
dose of luzopeptin E2 or a pharmaceutical composition thereof.
The following example serves to illustrate the
invention without in any way limiting it to the specific
embodiment disclosed. Pharmamedia is a trademark of Buckeye
Oilseed Products Co., Fort Worth, ~exas, for cottonseed meal.
Nutrisoy is a tradèmark of Archer Daniels Midland Co.,
Decatur, Illinois, for soybean meal ground into a flour.
Fermo 30 is a trademark of Yeast Products Inc., Clifton,
New Jersey, for a yeast powder. Skellysolve B is a trademark
lZZ~)7~;
of Skelly Oil Co., Kansas City, Missouri, for a petroleum
solvent comprising isomeric hexanes and having a boiling
point of 60-68C. Nylon Copol 8 is a tubular piece of
nylon used as a column in place of glass. The nylon can
withstand certain solv~nts and, when the chromatography
is completed, the column can be cut to obtain the fractiQns. ---
Example 1
Preparation of Luzopeptin E2
A. Fermentation
A well-grown vegetative preparation of
Actinomadura luzonensis strain G455-101 was maintained
frozen (-12C) in sucrose at a final concentration of
20~. This frozen vegetative preparation was used to
inoculate a vegetative medium containing 3~ cerelose, 1%
Pharmamedia (cottonseed meal), 1% Nutrisoy (soybean meal
powder) and 0.3% CaCO3. The seed culture was incubated
at 35C for 48 hours on a rotary shaker (210 rpm) and 7 ml
of growth was transferred to a 500 ml Erlenmeyer flask
containing 10~ ml of fermentation medium composed of 3%
cerelose, 2% Pharmamedia, 1% Fermo 30 (yeast powder),
0.5% CaCO3 and 0.15% DL-methionine, the pH being adjusted
to 7.0 before sterilization. Production of luzopeptin E2
reached a maximum between 72 and 96 hours at the 35C
incubat~on temperature.
B. Detection
-
Luzopeptin E2 can be isolated from the fermentation
mixture by dilution of the whole broth with an equal volume
of pH 8, 0.5M Tris-HCl buffer followed by an extraction with
two broth volumes of methylene chloride. After a brief
centrifugation to break the emulsion, the solvent layer was
-` ~Z207~6
spotted on Analtech silica gel plates and developed in a
xylene-methyl ethyl ketone-methanol (9:9:1) system. Luzopeptin
E2 appears at Rf 0.37, between BBM-928A (Rf 0.57) and BBM-928B
(Rf 0.27). All three compounds can be detected by W observa-
tion at 360 nm or by bioautography employing agar adjusted
to pH 8.0 and seeded with Bacillus subtilis ATCC 6633.
C. Isolation
The whole beer (10 1) from a fermentation of
Actinomadura luzonensis strain G455-101 grown in the
presence of DL-methionine (1.5 g/l), was adjusted to pH 8
with sodium carbonate and stirred for 2 hours with 4 1 of
n-butanol in the presence of diatomaceous earth as the
filter aid. The resulting mixture was filtered, and the
organic phase separated and concentrated under reduced
pressure to an oily residue. Trituration of the oily
residue with Skellysolve B (trademark of Skelly Oil Co.
for petroleum solvent comprising isomeric hexanes and
having a b.p. of 60-68C) and diethyl ether afforded,
after filtration and drying, 4 g of enriched luzopeptin
complex. The enriched luzopeptin complex was chromatographed
on a dry silica gel column prepared according to Loev and
Goodman (Progress in Separation and Purification, Vol. 3,
p. 73, 1970). Dry column silica gel (Merck; 280 g) was
deactivated by the addition of 40 ml of distilled water
and vigorous shaking for 3 hours followed by equilibration
for 3 additional hours with 18 ml of a mixture of xylene-
methyl ethyl ketone (1:1 by vol.). The silica gel was then
used to pack the column using a 160 gauge Nylon Copol 8
(4 x 100 cm) column with the help of glass wool.
The luzopeptin complex (4 g) was dissolved in
30 ml of xylene-methyl ethyl ketone (1:1 by vol), filtered,
coated on silica gel by removal of the solvent under reduced
pressure and applied to the dry column. The column was
developed with methyl ethyl ketone-xylene-methanol (5:5:1 by
vol) until the eluent had just overrun the bed of column.
lZZ0746
- 15 -
,
Observing the column with a portable W lamp at 360 nm, the
yellow fluorescing zones were cut and the resulting silica
gel sections eluted with chloroform-methanol (1:1 by vol)
and, after thin layer chromatography (TLC) analysis on
SiO2 (methyl ethyl ketone-xylene-methanol; 5:5:1 by vol),
the solids containing luzopeptin E2 were combined (494 mg).
The enriched luzopeptin E2 fraction (494 mg) was dissolved
in methylene chloride, coated on silica gel and chroma-
tographed on a 2 x 80 cm silica gel column slurry packed
in toluene with a toluene-methanol gradient (0 to 10~).
Fractions were collected and analyzed by TLC on silica gel
(methyl ethyl ketone-xylene-methanol; 5:5:1 by vol). Elution
with 7~ methanol yielded luzopeptin A and impurities followed
by elution of luzopeptin A and luzopeptin E2 (Fractions 71-75)
and pure luzopeptin E2 (Fractions 76-80). Crystallization
of pure luzopeptin E2 from methylene chloride-toluene
afforded 22 mg of crystalline luzopeptin ~2.
. .
