Note: Descriptions are shown in the official language in which they were submitted.
lZ~5at~7
(
BACKG~OUND ~)F THE INVENTION
(1) Field of the Invention
This invention relates to a new antitumor antibiotic
and to its production and recovery.
(2) Description of the Prior Art
The antitumor antibiotic of the present invention,
BBM-1644, is a new member of the protein antitumor antibiotics
exemplified by neocarzinostatin, macromomycin and auro~omycin.
Neocarzinostatin (also called zinostatin) is an acidic
protein macromolecule of molecular weight 10,700 c~nsisting of
a single polypeptide chain of 109 amino acids cross-linked by
two disulfide bridges. Production of neocarzinostatin by
fermentation of strains of Streptomyces carzinostaticus ~ar.
neocarzinostaticus is disclosed in U.S, P~tent 3,334,022 and
in _ Antibiotics 18: 68-76 (1965). The amino acid sequence
of neocarzinostatin is disclosed in Cancer Treatment Reviews
6: 239-~49 (1979).
Macromomycin is a neutral or weakiy acidic polypeptide
with an approximate molecular weight of about 15,00~. Production
of macromomycin by fermentation of Streptomyces macromomYceticus
(NIHJ MC-8-42) is disclosed in U.S. Patent 3,595,954 and in
J. Antibiotics 21: 44-49 (1968). Purification of macromomycin
-
and characterization data for the purified compound are disclosed
in J. Antibiotics 29: 415~423 (1976).
Auromomycin is a weakly acidic polypeptide with a
molecular weight of about 12,500 and an isoelectric point of
pH 5.4. It consists of 16 different amino acids. Isolation of
aur~momycin from the culture broth of Streptomyces macromomyceticus
and characterizing properties of the purified product are dis-
closed in J. Antibiotics 32: 330-339 (1979).
iZ15337
BBM-1644 may be differentiated from known polypeptide
antitumor antibiotics such as neocarzinostatin, macromomycin
and auromomycin by physico-chemical properties such as molecular
weight, amino acid content and paper electrophoresis.
SUMMARY OF THE INVENTION
There is provided by the present invention a new
protein antitumor antibiotic designated herein as BBM-1644,
said antibio~ic being prepared by culti~ating a new strain of
Actinomadura such as the one designated Actinomadura sp. strain
H710-49 (ATCC 39144) in an aqueous nutrient medium containing
assimilable sources of carbon and nitrogen under submerged aerobic
conditions until a surbstantial amount of BBM-1644 is produced
by said organism in said culture medium and optionally recovering
the BBM-1644 from the culture medium. The invention embraces
the BBM-1644 antibiotic in dilute solution, as a crude concen-
trate, as a crude solid and as a purified solid.
The ir.ventioll further relates to the antibiotic
~ BB.~-1644 which:
(a) is effective in inhibiting the growth of gram-positive
and acid-fast bacteria;
(b) is effective in inhibiting the growth of P388 leukemia in
mice;
(c) induces prophage in lysogenic bacteria;;
(d) is soluble in water but practically insoluble in methanol,
ethanol, acetone, ethyl acetate and n-hexane;
(e) exhibits an infrared absorption spectrum (KBr) substantially
as shown in FIG. 1;
tf) exhibits ultraviolet absorption spectra in water, OoOlN
HCl and 0.0lN naOH substantially as shown in FIG 2;
(g) has an optical rotation of [a~D -75.6 in 0.25~ aqueous
solution;
,
~2~S337
-3a-
(h) has no definite melting point but gradually decomposes
above about 240C;
(i) moves about 8.7 cm toward the anode during paper electro-
phoresis at 4500 VO for 1 hour using 0.05M barkital buffer of
pH 8.6;
(j) has the following elemental analysis: C, 46.60%; H, 6.45%;
N, 13.34%; S, 0.20% and O (by difference), 33.41%;
(k) is a high molecular weight peptide for which a molecular
weight of about 22,000 is indicated;
(1) decolorizes potassium permanganate solution and gives
positive Folin-Lowry, xanthoprotein, biuret and ninhydrin
reactions and negati~e anthrone and Sakaguchi reactions; and
(m) gives by hydrolysis the following relative amino acid
composition based on the content of leucine being arbitrarily
assigned as l o O alanine (8.8), aspartic acid (6.0),
half-cystine (1.0), glutamic acid (5.7), glycine (8.7),
isoleucine (2.3), leucine (1.0), phenylalanine (1.4?, proline
(4.1), serine (1.6), threonine (7.2), tyrosine (0.6) and valine
( 1 1 0 1 ) O
The process for the production of the antibiotic,
BBM-1644, which comprises cultivating a BBM-1644-producing
strain of Actinomadura spO in an aqueous nutrient medium
containing assimilable sources of carbon and nitrogen under
The invention still further relates to a process for
the production for of the antibiotic BBM-1644, which
comprises cultivating a BBM-1644-producing strain of
Actinomadura sp. in an aqueous nutrient medium containing
assimilable sources of carbon and nitrogen under submerged
aerobic conditions until a substantial amount of BBM-1644
is produced by said organism in said culture medium.
37
-3b-
DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the infrared absorption spectrum of BBM-1644
(KBr pellet).
FIG. 2 shows the ultraviolet absorption spectra of BBM-1644
in water, 0.0lN BCl and 0.0lN NaOH.
DETAILED DESCRIPTION
This invention relates to a novel protein antitumor
antibiotic designated herein as BBM-1644 and to its preparation
by fermentation of a new strain of Actinomadura designated
, ~
- ` ` ~2~S337
A'ctinomadura sp. strain H710-49. The above organism was
isolated from a soil sample collected in West Germany. A
biological,ly pure culture of the organism has been deposited
with the American Type Culture Collection, Washington, D.C.,
and added to its permanent collection of microorganisms as
ATCC 39144.
BBM-1644 inhibits the growth of various gram-
positive and acid-fast bacteria. The antibiotic also exhibits
phage inducing pxoperties in lysogenic bacteria and inhibits
growth of lymphatic and solid tumors such as P388 leukemia in
mice. The new antibiotic, therefore, may be used as an anti-
bacterial agent or as an antitumor agent for inhibiting
mammalian tumors. '
The'Microorganism
The actinomycete strain No. H710-49 was isolated
from a soil sample and prepared by conventional procedures as
a biologically pure culture for characterization. Strain
H710-49 forms both substrate and aerial mycelia. The substrate
mycelium is long, branched and not fragmented into short
filaments. Short spore-chains are born on the tip or monopodial
branch of aerial mycelium. The spore-chains contain 2--'to i~''s'po'res
in a chain (mostly i to' 4-spores) and are straight, hooked or lo'oped
in shape. The spores have a warty surface and are oval to
elliptical (O.5~0.6 x O.7~1.2~m) in shape with a round or
p,ointed end. Mature spores are often separated by empty hyphae.
Terminal swellings of hyphae are occasionally observed on the
substrate mycelium in Czapek's agar and Bennett's agar. Motile
spores, sporangia or sclerotic granules are not seen in any media
examined.
~;~i5;~37
-s Unlike ordinary species of the genus Streptomyces,
strain H710-49 grows slowly and forms poor aerial mycelium
in chemically defined media and natural organic media. The
color of aerial mycelium is white and turns to a pinkish
shade after sporulation in oatmeal agar, inorganic salts-
starch agar and glycerol-asparagine agar. Mass color of
the substrate mycelium is colorless, yellow, reddish brown
or dark grayish brown. Melanoid pigment is not produced,
but a lemon yellow diffusible pigment is seen in glycerol-
asparagine agar, tyrosine agar and Pridham-Gottlieb's basal
agar supplemented with any one of glycerol, L-arabinose,
D-xylose, L-rhamnose, D-glucose, D-fructose, trehalose and
D-mannitol. Strain H710-49 grows at 20C, 28C and 37C,
but not at 10C or 41C. It is sensitive to NaCl at 10%
but not at 7~, and resistant to lysozyme at 0.001%,
D-Galactose, D-mannose, sucrose, raffinose and inositol
are not utilized by the strain. Cultural and physiological
characteristics of strain H710-49 are shown in Tables 1 and
2, respectively. The pattern of carbon source utilization by
the strain is shown in Table 3.
Table 1
Cultural characteristics* of strain H710-49
_
Tryptone-yeast extract G** poor to moderate; floccose,
broth (ISP No. 1) sedimented and not pigmented.
Sucrose-nitrate agar G scant
(Czapek's agar) R colorless to pale orange
yellow (73)***
A scant; white (263)
D none
l~lS~37
-- 6 --
Table 1 - continued
_
Glucose-asparagine agar G poor
R yellowish white (92) to deep
orange yellow (69)
A very scant; white (263)
D none
Glycerol-asparagine agar G poor to moderate
(ISP No. 5~ R pale yellow (89) to dark
orange yellow (72)
A poor; white (263) to pale
yellowish pink (31)
D brilliant yellow (83)
Inorganic salts-starch G poor to moderate
agar (ISP No. 4). R colorless to deep yellow (85)
A poor; pinkish white (9) to
pale yellowish pink (31)
D none
~yrosine agar (ISP No. 7) G moderate
R brownish orange (54) to
moderate reddish brown (43)
A poor; white (263) to pale
yellow (89)
D strong yellow (84)
Nutrient agar G poor to moderate
R yellowish white (92) to
moderate yellowish brown (77)
A poor; white (263)
D none
i~7
Table 1 continued
Yeast extract-malt extract G moderate
agar (ISP No. 2) R dark yellow (88) to dark
brown ~59)
A scant; white (263)
D light olive brown (94)
Oatmeal agar (ISP No. 3) G poor
R colorless
A poor; white (263) to pinkish
white (9)
D none
Bennett's agar G moderate
R grayish yellowish brown (80)
to dark grayish brown (62)
A very scant; white (263)
D moderate olive brown (95)
Peptone-yeast extract- G poor
iron agar (ISP No. 6) R grayish yellow (90) to dark
grayish brown (62)
A poor; white (~63)
D none to moderate yellowish
brown (77)
* observed after incubation at 28~C for 3 weeks
** Abbreviation: G-- Growth; R - Reverse color; A - Aerial myc~lium;
D - Diffusible pigment
*** Color and number in parenthesis follow the color standard in
"Kelly, K. L. & D. B, Judd: ISCC-NBS color-name charts illustrated
with Centroid Colors. U.S. Dept. o~ Comm. Circ. 553, Washington,
D.C., Nov., lg75"
12~5337
--: ~ V ~ h tl) 1:: 0 U U
E tn ~ ~I X~ ~ I a) ,4 X X X
:~ . I ~ ~ X ~ u~ a~ q) a
,~ aJ ~15 EO a~ u~ O
O U ~ ~ ~ .
E~ tJ~ ,a tO 0
F ~ O
l ~ ~ h aJ tJ) rl
~n ~ s o ~ ~ ~ ,Y u ~ o o
O ~) O :~ U O ~ S O .C
.C ~ V rl ~ U a, ~ ~ ~: ~ ~ Ll Q~
a~ ~ ~ O ~.
~ Q) a~ 1 N ~1 X
l ~: m ~ a E~ R
o
_l
I~
:~: .
~ C~ o
.,., o o ~ '
CC~ N S ~ ~ ~
Ll ~ ~ N 1: 0 ra
3 ~ rl ~1
u~ ~ a~ O C~ ~: ~ S
1~ 0 tl~ ~ ~
S tJ~ 3 ,4
O S ~ ~ R O
~ 3 C~ Q) S-/ O
U~ ~ O Z O .
_I lQ ~ ~ C~ ~ a) N ~ a) ~
R ~1 u~ _~ ~ o 1~ ~ O ~ ~ ~ -
~ ~ ~ ~ ~ ~ ~1 0 ~ S dP ~ o'P 5
E~ a) O E~ ~ ~ a) O U ~ , U h ~ o tq _l
~ J C 3 ~0 3
~ u~ X ~ ~a O t~1 ''O ~ E~ ~ ~a ~ o tn o O o
~ O a o ~ o ~1~ o o o a) o h ~O S~
~1 ~ ~ Z C) ~ o t~ u~
S
O
_I
U
.,1
o
_I .Y
O h _I
~t O ,1
0
:~
.C
~' O ~ O
u~ E
~1 0
Z
~ U O
E :~ O
o
~u S ~ O
O ~ O ,~
r~ S ~1
.IJ ~ U) N
t~) 3 ~
u~ ~ o
~ ~ O ~
E~ ~Y; ~ ~ u~ P; h Z
~;~15337
Table 3
Utilization of caxbon sources* by strain H710-49
Glycerol +
D(-)-Arabi~ose
L(+)-Arabinose +
D-Xylose +
D-Ribose
L-Rhamnose +
D-Glucose +
D-Galactose
D-Fructose +
D-Mannose
L(-)-Sorbose
Sucrose
Lactose
Cellobiose +
Melibiose
Trehalose +
Raffinose
D(+)-Melezitose
Soluble starch +
Cellulo~e - -
Dulcitol
Inositol
D-Mannitol +
D-Sorbitol
Salicin
* Observed after incubation at 28C for 3 weeks.
Basal medium: Pridham-Gottlieb inorganic medium
~:~lS337
--10 --
Purified cell-wall of strain H710-49 contains meso-
dial[linopimelic acid but lacks glycine. The whole ce11 hydroly-
zate sl~ows the presence of madurose (3-0-methyl-D-galactt~se),
glucose, ribose and a small amount of mannose. The cell-wall
composition and whole cell sugar components of strain H710-49
indicate that the strain belongs to cell-wall Type IIIB.
The above-described characteristics of strain H710-49
resemble those of the genus Actinomadura. According to the
numerical taxonomy of Actinomadura and related actinomycetes
by Goodfellow et al. in J. Gen. Microbiol.~112 95-111 (1979),
mos~ Actinomadura species of soil origin are classified into
Cluster No. 7 among the 14 clusters described. Strain No.
H710-49 is most related $o the species of Cluster 7. Nonomura
and Ohara in J. Ferment. Technol. 49: 904-912 (1971) reported
-
five saprophytic species of the genus Actinomadura and t~onomura
[_ Ferment. Technol. 52: 71-77 (1974)] and Preobrazhenskaya et
al. ~Actinomycetes and Related Organisms _: 30-38 (1977)]
described the identification and classification of Actinomadura
species. As a result of comparison with known Actinomadura
species described in the literature, strain H710-49 is considered
to belong to a new species o E Actinomadura similar to A. roseola,
A. salmonea, A. ~rinacea or A. corallina described in the
Preobrazhenskaya et al. reference above and in Japanese Kokai
55/94391.
It is to be understood that for the production of
BBM-1644, the present invention, though described in detail with
reference to the particular strain Actinomadura sp. strain H710-49
(ATCC 39144 ), is not limited to this microorganism or to micro-
organisms fully described by the cultural characteristics dis-
closed herein. It is specifically intended that the invention
embrace strain H710-49 and all natural and artificial BBM-1644-
producing variants and mutants thereof,
~2~5337
-~; Antibiotic Production ~
The BBM-1644 antibiotic of the present invention
may be prepared by cultivating a BBM-1644-producing strain
of the genus Actinomadura, preferably a strain of Actinomadura
sp. having the identifying characteristics of ATCC 39144
or a mutant thereof, in a conventional aqueous nutrient medium.
The organism is grown in a nutrient medium containing known
nutritional sources for actinomycetes, i.e. assimilable sources
of carbon and nitrogen plus optional inorganic salts and other
known growth factors. Submerged aerobic conditions are
preferably employed for the production of large quantities of
antibiotic, although for production of limited amounts, surface
cultures and bottles may also be used. The general procedures
used for the cultivation of other actinomycetes are applicable
to the present invention.
The nutrient medium should contain an appropriate
assimilable carbon source such as glycerol, L(+)-arabinose,
D-xylose, D-ribose, D-glucose, D-fructose, soluble starch,
D-mannitol or cellobiose. As nitrogen sources, ammonium chloride,
ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc.
may be used either alone or in combination with organic nitrogen
sources such as peptone, meat extract, yeast extract, corn steep
liguor, soybean powder, cotton seed flour, etc. There may also
be added if necessary nutrient inorganic salts to provide
sources of sodium, potassium, calcium, ammonium, phosphate,
s~ulfate, chloride, bromide, carbonate, zinc, magnesium, manganese,
cobalt, iron, and the like.
Production of the BBM-1644 antibiotic can be effected
at any temperature conduci~e to satisfactory growth of the
producing organism, e.g. 20-37C, and is conveniently carried
out at a temperature of around 27-32C Ordinarily, optimum
production is obtained in shaker flasks after incubation periods
of about 6-7 days~ When tank fermentation is to be carried out,
.
lZ~S337
- 12 -
it is desirable to produce a vegetative inoculum in a nutrien~
~roth by inoculating the broth culture with a slant or soil
culture or a lyophilized culture of the organism. After
obtaining ~n active inoculum in this manner, it is transferrea
aseptically to the fermentation tank medium. Antibiotic
production may be monitored by the paper disc-agar diffusion
assay using Bacillus subtilis M45 ~Rec mutant; Mutation Res.
16: 165-174 (1972)~ as the test organism.
.
Isolation and Purificati~n
.
When fermentation is complete, BBM-1644 exists mainly
in the liquid part of the fermented broth after separation of
the solid part by filtration or centrifugation. Thus, the
harvested broth may be ~eparated into mycelial cake and broth
supernatant by centrifugation. The filtrate is then concentrated
and dialyzed against tap water by a semipermeable membrane such
as a cellophane tube to remove permeable impurities. The inside-
retained solution (after remoYal of insoluble materials) contain-
ing the BBM-1644 may then be saturated with a salting out reagent
such as æmmonium sulfate to precipitate out BBM-1644 as a crude
solia. This solid may be dissolved in water and desalted by
dialysis against tap water.
Further purification of the crude BBM-1644 may be
accomplished by conventional procedures used with other acidic
polypeptides. For example, the aqueous solution containing
BBM-1644 may be adsorbed on an ion exchanger such as DEAE-
5ephadex, DEAE-Cellulose* CM-Sephadex*or CM-cellulose* and eluted
with a neutral ~alt solution. Successive chromatographic steps
ar~ pre~erably employed with a gradient concentration of salt
solution used as the eluant. Aqueous fractions containing the
purified BBM-1644 axe then concentrated to dryness as by
lyophilization.
*Trade Mark
LS~37
- 13 -
.
- Physico-chemical'Properties of BBM-1644
BBM-1644 is isolated as an amorphous white powder
up~n lyophilizationO When examined by high voltage paper
electrophoresis (4500 V in 0.05M-bar~ital buffer at pH 8.6~,
~BM-1644 migrates as an acid travelling 8.7 cm toward the
anode after one hour. BBM-1644 does not show a definite
melting point and gradually decomposes above 240C. It is
soluble in water, but practically insoluble-in common organic
sol~ents such as methanol, ethanol, acetone, ethyl acetate and
n-hexane. The antibiotic shows an optical rotation of ~j26=
-7~.6 in 0.25% aqueous solution.--As depictéd'in ~IG. 2, the
W spectrum of B8M-1644 shows absorption maxima at 275 nm
(ElCm 8.2) and 310 nm (ElCm 4.6, shoulder) in aqueous solution.
It exhibits a nearly identical W spectrum in water and in
o.oiN HCl solution, but only a single maximum at ~B5 nm (El%m 8.9)
in 0.01N NaOH solution. The IR spectrum of BBM-1644 measured
in KBr is shown in FI G . 1. The spectrum indicates the
presence of NH and OH groups (3300~2980 cm 1) and amide groups
(1650 and 1540 cm 1). The antibiotic gives positive reactions
to Folin-L~wry, xanth~protein, biuret and ninhydrin reagents and
de~olorizes potassium permanganate solution. It is negative
to anthrone and Sakaguchi reactions. When co-chroma't'o'g'raphëd
on Sephadex G,-75 with o~albumin (MW 43,000), chymotrypsinogen
(25,000) and ribonuclease A (13,700), BBM-1644 is eluted just
after chymotrypsinogen and therefore its molecular weight is
~stima~ed to be hround 22,000. Elemental analysis of BBM-1644
indicates carbon 46.60~, hydrogen 6.45%, nitrogen 13.34% and
sulfur 0.20~. The presence o~ 13 kinds of amin~ acids in the
BBM-1644 molecule was re~ealed by amino acid analysis as shown
in Table 4. asi~ amino acids such as lysine, histidine and
arginine are not present in BBM-1644.
*Trade Mark
~.
~5337
Table 4
:: Amino acid comeosition of BBM-1644
Relative composition*
Amino acid of amino acids
Alanine 8,8
Aspartic acid 6.0
half-Cystine 1.0
Glutamic acid 5.7
Glycin 8,7
Isoleucine 2.3
Leucine 1.0
Phenylalanine 1.4
Proline 4.1
Serine 1.6
Threonine . 7.2
Tyrosine O.6
Valine 11.1
* Content of leucine was arbitrarily assigned as 1Ø
~Z~LS33~
BBM-1644 is fairly stable in the pH range of 2~9,
b~t the stability declines sharply beyond this pH r~nge.
The aqueous solution of BBM-1644 is stable for 2 hours at
50C at neutral pH. Upon exposure to ultraviolet li~ht,
the antibiotic activity of BBM-1644 is lost within 20 minutes.
The physico-chemical properties of BBM-1644 described
above indicate that it is a member of the protein antitumor
antibiotic group which includes neocarzinostatin,~macromomycin
and auromomycin. BBM-1644, however, can be differentiated
from the known protein antitumor antibiotics by its molecular
weight, amino acid content and paper electrophoresis, The
paper electrophoretic mobilities of BBM-1644, neocarzinostatin
and macromomycin are shown in Table 5.
Table 5
Paper ele_trophoresis*
Mobility (mm from the ~oadinq spot)
BBM-1644 +87
Neocarzinostatin ~31
Macromomycin -20
* 4,500 V, 1 hour; Barbital buffer p~ 8.6
The neocarzinostatin group of antibiotics commonly exhibit two
W absorption maxima at around 275 and 350 nm, while BBM-1644
has the W maxima at around 275 and 310 nm. It has recently
been reported in Biochem. Res. Commun. 95: 1351-1356 (1980)
_
that the maximum at 350 nm of the neocarzinostatin antibiotics
might be due to the non-protein chromophores which are essential
for their'biological activity. It is noted that BBM-1644 has
a chromophore different from that of the known ne~carzinostatin
group of antibiotics.
~ S~37
- 16 -
Biological Properties of BBM-1644
.,- .
The antibacterial activity of BBM-1644 was determined
by the serial two-fold agar dilution method. Nutrient agar
medium was used for gram-positive and gram-negative bacteria;
nutrient agar medium containing 4% glycerol for acid-fast
bacteria and Sabouraud agar medium for fungi. The activity
was expressed as minimum inhibitory concentration (MIC~ in
the agar medium and the results are shown in Table 6 along
with those of neocarzinostatin. BBM-1644 showed p~tent inhibitory
activity against gram-positive and acid-fast bacteria but did
not inhibit the growth of gram-negative-bacteria and fungi,
The antibacterial spectrum of BBM-1644 is similar to that of
neocarzinostatin, while the intrinsic activity of BBM-1644
is more potent than the latter in some o~ the test organisms.
Table 6
In vitro antimicrobial activity
MIC in mcq/ml
Test organismBBM-1644 Neocarzinostatin
StaPhylococcus aureus FDA 209P 0.4 1.6
Staphylococcus aureus Smith 0.2 0.8
-
Streptococcus pyogenes A20201 6.3 3.1
Micrococcus luteus PCI 1001 1.6 1.6
Micrococcus flavus D12 1.6 1.6
Bacillus subtilis PCI 219 0.8 3.1
Escherichia coli NIHJ >100 >100
Klebsiella pneumoniae D-ll >100 >100
Proteus vulqaris A9436 >100 >100
Pseudomonas aeru~inosa A9930 >100 >100
Mycobacterium smegmatis 607 D8712.5 50
Mycobacterium phlei D88 3.1 12.5
Candida albicans IAM 4888 ~100 >100
~215337
The ability of BBM-1644 to induce prophAge--~n
lys~genic bacterium (I~B) w~s determined by the method
o~ ~ein et.al. (Nature 196: ~83-7B4, 1962) using neo-
carzinostatin as a reference compound. The plaque count
was made on agar plates containing test material (T) and
control (C). A ~/C ratio of the plaque counts of greater
than 3 was considered significant and the ILB activity was
expressed by the minimum inducing concentration of the test
compound. As shown in Table 7, the ILB activity of BBM-1644
is similar ~o that observed with neocarzinostatin, the
minimum inducing concentration being 1 mcg/ml.
Table 7
Induction of lysogenic ~acterium bY BBM-1644
I~B activitY (T/C)*
100 10 1 0.1 mcq/ml
_
BBM-1644 10.4 10.3 6.0 1.2
Neocarzinostatin 28.6 20.4 10.8 0.7
* significant activity: T/C O~ ~3 !
The antitumor acti~ity of BBM-1644 was determined in
mi~e (BDFl strain) against lym~hocytic leukemia P388. Each mouse
was inoculated intraperitoneally with 3 x 105 cells of the ~umor.
Graded doses of the antibi~tic were administered t~ mice intr~-
peritcneally 24 hours a~ter tumor implantation. The treatments
were given on~e a day on days 1.4 ~nd 7 (q3d x3 schedule~ or
9 consecutive days ~qd 1~9 schedule). Neocarzinostatin was
comparatively tested as ~ reference antitumor agent and the
results are summarized in Ta~le 8. BBM-1644 was highly active
against the mouse leukemia in a dose range of 0.03~1~0 mg/kg/day
.
~i5337
- 18 -
by both treatments. The antitumor activity of BBM-1644 was
about the same as (qd 1~9 schedule~ or 3 times more potent
(q3d x 3 s~chedule) than that of neocarzinostatin in terms of
minimum effective dose.
Table 8
Antitumor activity aqainst leukemia P388
.
T/C (%) in MST*
Dose in mg/k~/day, ip, q3d x 3
1 0.3 0.1 0.03 0.01
BBM-1644 188 188 - 138 125 113
Neocarzinostatin -163 15~ --125- 113
T/C (%) in MST
Dose in mg/kg/day, ip, qd 1~9
0.3 0.1 0.03 0.01
BBM-1644 125~ 5 ;138 - 113
Neocarzinostatin 163- 138 ----125--~ 113
.
* median survival time;-, signi-ficant activity: T/C of _125%
Antitumor activity of B~M-1644 was also indicated by a
second test against P388 leukemia in mice, the results of which
are shown below in Table 9. Details of the methods used in
this test have been described in Cancer Chemother. Rep. 3: 1-87
tPart 3), 1972.
~2~S;~3~
-- 19 --
Table 9
Effect of BBM-1644 on P388 Leukemla
Effect AWC
Treatment Dose, IP MST MST sm Survivors
Material Schedule mg/kg/inj Days %T/C d.6 Day 5(303
Neocarzino- D.1,4&7 1.6 8.0 89 -4.8 6/6
statin 0.8 10.5 117 -4.3 6/6
0.4 15.5 172 -3.2 6/6
0.2 15.0 167 -2.5 6/6
0.1 13.5 150 -1.2 6/6
0.05 12.0 133 -1.7 6/6
BBM-1644 D.l 2.56 9.0 100 - 4/6
1.28 14.0 156 -4.1 6/6
0.64 14.5 161 -4.9 6/6
0.32 16.5 183 -4.8 6/6
0.16 14.0 156 -4.1 6/6
0.08 12.0 133 -2.g 6/6
0.04 10.5 117 -2.7 6/6
0.02 11.0 122 -2.5 6/6
D.1,4&7 1.28 23.5 261 -3.3 6/6
0.64 19Ø 211 -3.5 6/6
0.32 18.5 206 -4.3 6/6
0.16 14.5 161 -3.8 6/6
0.08 12.0 133 -3.3 6/6
0.04 12.0 133 -2.9 6/6
0.02 10.0 111 -2.5 6/6
0.01 10.0 111 -1.9 6/6
QD 1~9 0.64 8.0 89 -5.2 6/6
0.32 10.0 111 -4.2 6/6
0.16 19.0 211 -4.3 6/6
0.08 18.0 200 -4.2 6/6
0.04 15.5 172 -3.5 6/6
0.02 13.0 144 -3.3 6/6
~ 0.01 12.0 133 -2.2 6/6
0.00511.0 122 -1.1 ~/6
~S;~37
~ 20 -
Table 9 continued
Effect A~C
Treatment Dose, IP MST MST gm Survivors
Material _Schedule mg/k~/ini Days ~T/C d.6 Day 5(30)
.
Control~107 Saline 8.0 - - 10/10
6 Saline-` 9.0 - -0.3 20/20
105 Saline 11.0 - _ 10/10
104 Saline 14.0 - _ 10/10
.
Tumor inoculum: 106 ascites cells, ip ~plus titration)
Host : CDFl ~ mice.
Tox : <4/6 mice ali~e on Day 5.
Evaluation : MST = median survival time,
Effect : % T/C = (MST treated/MST control) x 100.
Criteria : % T/C _ 125 considered significant antitumor
activity.
The acute toxicity of BBM-1644 was determined in mice
(dd Y strain) by single intraperitoneal administration, the L~50
being calculated as 5.8 mg/kg.
As shown above, BBM-1644 possesses potent antibacterial
activity against gram-positive and acid-fast bacteria and is thus
useful in ~he therapeutic treatment of mammals and other animals
for infectious diseases caused by such bacteria. Additionally,
it may be utilized for other conventional applications of anti-
bacterial agents such as disinfecting medical and dental equipment.
The induction of prophage in lysogenic bacteria and the
marked antitumor activity shown against P388 leukemia in mice
indicate that BBM-lÇ44 is also therapeutically useful in inhibiting
the growth of mammalian tumors.
The present invention, therefore, provides a method for
therapeutically treating an animal host affected by a bacterial
infection or by a malignant tumor which comprises administering
~iL5;~37
- 21 -
to said host an effective antibacterial or tumor-inhibiting
dose of BBM-1644 or a pharmaceutical composition thereof.
~ n another aspect, the present in~ention provides
a pharmace.utical composition which comprises an effective
antibacterial or tumor-inhibiting amount of BBM-1644 in
combination with an inert pharmaceutically acceptable carrier
or diluent. These compcsitions may be made up in any pharma-
ceutical form appropriate for parenteral administrati~n.
Preparations according to the invention for parenteral
administration include sterile aqueous or non-aqueous 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.
It will be appreciated that the actual preferred
amounts of the BBM-1644 antibiotic used will vary according to
the particular composition formlllated, the mode of application
and the particular situs, host and disease being treated,
Many factors that modify the action of ~he drug will be taken
into account by those skilled in the art, for example, age,
body weight, sex, diet, time o~ administration, route of
administration, rate of excretion, condition of the host, drug
combinations, reaction sensitivities and severity of the disease.
Administration can be carried out continuously or periodically
within the maximum tolerated dose. Optimal application rates
for a given set of conditions can be ascertained by those skilled
in the art using conventional dosage determination tests in
view of the above guidelines.
The following examples are pro~ided for illustratiYe
purposes only and are not intended to limit the scope of the
invention. DEAE Cellulose is a diethylam'noethyl ion exchange
cellulose. SEPHADEX G-50 is a filtration gel manufactured by
Pharmacia Fine Chemicals, Inc. DEAE SEPHADEX A-S0 is a diethyl-
aminoethyl anion exchange gel manufactured by Pharmacia Fine
5337
- 22 -
~ ,
Chemicals, Inc. SEPHADEX is a trademark of Pharmacia Fine
Chemicals, Inc. -
EXAMPLE 1
Fermentation of BBM-1644
An agar slant with-well-established growth of Actinoma-
d _ sp'.'H710-49-was u'sed to inoculate seed mediu~ (100 ml in a
500-ml Erlenmyer flask) containing 1% mannitol, 2% peptone and
1~ yeast extract, the pH being adjusted to 7.2 before steriliza-
tion. The seed culture was incubated at 32C for 72 hours on
a rotary shaker (250 rpm) and 5 ml of the culture was transferred
to the second seed medium (100 ml) composed of the same
composition as the fi'rst~seed medium.~~~'It was cultiYated
under the same condition as that used for the first seed culture.
Five ml of the inoculum growth thus prepared was employed to
start fermentation in ~00-ml Erlenmyer flasks which contained
100 ml of fermentation medium having composition of 2.5% mannitol,
O.5% glucose, 1% soybean meal, 0.5~ peptone, 1% meat extract,
0.3~ CaCO3 and 0.2~ NaCl. Fermentation was carried out at 28C
on a rotary shaker with 250 rpm rotation. The antibiotic
production was monitored by the paper disc-agar diffusion assay
using Bacillus subtilis M4~ (Rec mutant) as the test organism.
. _
The antibiotic activity in the culture broth gradually increased
with the progress of fermentation and reached about 300 mcg/ml
after 6~7 days.
. . .
EXAMPLE 2
~ .
The harves~ed broth (18 liters~ obtained from the
Example 1 fermentation was separated to mycelial cake and broth
supernatant by using a sharpless centrifuge apparatus ~Kokusan
No. 4~). The filtrate was concentrated below 40C to one-tenth
~2153:~7
- 23 -
the original volume and the concentrate was dialyzed by
c~ phane tubing (Union Car~ide) against tap water ln a
cold room. The inside-retained solution was concentrated
to about 1.5 liters which was centrifuged (8,000 G) to remove
insoluble materials. The clear supernatant was saturated
with ammonium sulfate and allowed to stand for 5 hours at
5C. The precipitate formed was collected by centrifugation,
dissolved in water (300 ml) and desalted by dialysis against
tap water. The dialyzed solution (700 ml) contained 22 grams
of crude solid of BBM-1644 as revealed by lyophilization of
a part of the solution. The rest of the solution was used for
subsequent purification without concentration in order to avoid
decomposition. The antibiotic solution was passed through a
column of DEAE-cellulose (Cl , 400 ml) and the column was washed
with water (1 liter) and developed with 1/15M phosphate buffer
(pH 7.0) containing 0.3M sodium chloride. The active fractions
were combined (300 ml), dialyzed for 18 hours against tap watex
and chromatographed on a column of DEAE-cellulose (400 ml) which
had been equilibrated with l/lSM phosphate buffer (pH 7.5).
The column was developed with the same buffer solution containing
an increasing amount of sodium chloride (0~0.2M). The active
eluate was desalted by dialysis and charged on a column of
DEAE-Sephadex A-50 (17 ml). The column was developed with 1/15M
phosphate buffer (pH 7.5) containing a gradient concentration of
sodium chloride (0~0.3M). The active fractions as determined by
B. subtilis M45 assay were pooled and dialyzed against running
water for 18 hours. The desalted solution was chromatographed
on a column of DEAE-Sephadex A-50 (18 ml) using a 1/15M phosphate
buffer (pH 7.0)-NaCl (0~0.3M) system as an eluant. The
appropriate fractions were collected, concentrated to 10 ml and
applied on a column of Sephadex G-50 for desalting. The column
was eluted by deionized water and the active eluate lyophilized
to afford 120 mg of white powder. The sample of BBM-1644 thus
obtained was homogeneous as revealed ~y polyacrylamide gel
electrophoresis.
