Note: Descriptions are shown in the official language in which they were submitted.
Back~round of the Invention
This invention relates to chemical compounds produced by fermen-
tation and used to inhibit the growth of leukemia cells.
10~674~
Summary of the Invention
This invention relates to two new antibiotics
which each exhibit a high activity inhibitory to the gro~th
of leukemia cells and are useful as anti-tumor agents but
exhibit low antibacterial activity. More particularly, this
invention relates to the new antibiotics neothramycin A and
neothramycin B, (initially designated MC916-A substance and
MC916-B substance), respectively, and also to a process for
the production of these new antibiotics by cultivation of
a strain of Streptomyces. This invention also relates to the
recovery and purification of these specific new antibiotic
substances and to their use for pharmaceutical purposes.
Hereinafter, by the term "neothramycin" is meant
neothramycin ~. and neothramycin B or their mixture unless
otherwise stated.
Some antibiotics which are useful as anti-tumor
agents for the therapeutic treatment of leukemia, for example,
are daunomycin, adriamycin, etc. In an attempt to obtain
further new anti-tumor agents of antibiotic type, we collected
various soil samples, isolated microorganism frcm such soil
samples and investigated metabolic products which are produced
by the aerohic cultivation of the isolated microorganisms.
We isolated a new microorganism from a soil sample collected
in the grounds of Biseibutsu Kagaku Kenkyu-sho in
Shinagawa-~u, Tokyo, Japan, and we have designated this
newly isolated microorganism as MC916-C4 strain. It has
been confirmed that this ~lC916-C4 strain belongs to the
genus Streptomyces. We have now found that two new anii-
biotics having a low antibacterial activity but high
activity inhibitory to the growth of leukemia L-1210 cells
1056747
in mice and to the growth of a certain kind of tumor cells
are produced and accumulated in the culture broth of the
MC916-C4 strain. T~e have now succeeded in isolating these
new antibiotics from the culture broth and designated them
as neothramycin A and neothramycin B, respectively.
An object of this invention is to provide new
substances which are useful as anti-tumor agents. Another
object of this invention is to provide neothramycin A and
neothramycin B, either alone or in mixture thereof as new
and useful anti-tumor agents. A further object of this inven-
tion is to provide a process for the preparation of neo-
thramycin A and neothramycin B by cultivation of the MC916-C4
strain. Other objects of this invention will be clear from
the following descriptions.
According to one aspect of this invention, there
is provided as a new antibiotic substance, neothramycin
substance having an activity inhibitory to the growth of
leukemia L-1210 cells in mice and a low antibacterial
activity, said substance having an acidic function; being
soluble in methanol, ethanol, propanol, chloroform and dioxane
and slightly soluble in water but sparingly soluble or sub-
stantially insoluble in ethyl ether and n-hexane; being
positive to ~ydon-Smith reaction and red tetrazolium reaction,
weakly positive to ninhydrin reaction but negative to Ehrlich
reaction and Sakaguchi reaction; giving essentially only
carbon, hydrogen, nitrogen and oxygen upon elemental analysis
thereof; exhibiting a relative mobility of said substance
to alanine (1.0) being 0.17 on high-voltage filter paper
electrophoresis ~3500 volts, 35 minutes) using formic acid-
acetic acid-water t25:75:900 by volune) as an electrolyte
;~,
1056747
solution; said substance being at least one member selected
from the group consisting of neothramycin A and neothramycin
B;
a) said neothramycin A being further characterized
by giving C, 57.46%, H, 5.76%, N, 9.84~ and the remainder
oxygen upon elemental analysis thereof; giving a molecular
weight of 250 to 300 as measured by ~arger - AXiya method;
having an infrared absorption spectrum pelleted in potassium
bromide characterized by absorption peaks at 3450, 2750, 1630
(shoulder), 1600, 1510, 1460, 1440, 1410, 1280, 1200, 1180,
1120, 1080, 1010, 870, 790 and 760 cm 1; having ultraviolet
absorption spectra characterized by absorption maxima at
223 nm (El%Cm 855), 240 nm (shoul~er), 265 nm (El%Cm 290)
and 318 nm (El~Cm 156) in a solution thereof in 10~ water-
methanol, by absorption maxima at 223 nm (El%Cm 885),
240 nm (shoulder), 265 nm (~l%cm 290) and 320 nm (El%Cm 139)
in a solution thereof in N/10 HCl-methanol (1:9) and by
absorption maxima at ~28 nm (El%Cm 635~, 254 nm (El cm 566),
291 nm (El~Cm 422) and 324 nm (El%Cm 412) in a solution
thereof in N/10 NaOH-methanol (1:9); and giving an Rf value
of 0.57 in thin layer chromatography on silica gel with
chloroform-methanol (10:1 by volume) as the developing
solvent; and
b) said neothramycin B being further characterized by
giving C, 57.00%, H, 5.58%, N, 9.75% and the remainder
oxygen upon elemental analysis thereof; giving a molecular
weight of 250 to 300 as measured by ~arger - Akiya method;
having an in rared absorption spectrum pelleted in potassium
bromide characterized by absorption peaks at 3400, 2960,
1630 (shoulder), 1600, 1510, 1440, 1400, 1280, 1200, 1120,
- 4 -
.
~056747
1080, 1010, 990, 940, 870, 790 and 760 cm 1; having ultra-
violet absorption spectra characterized by absorption maxima
at 224 nm (El~Cm 935), 240 nm (shoulder), 265 nm (shoulder)
and 318 nm (El~Cm 167) in a solution thereof in 10~ water-
methanol (1:9), by absorption maxima at 224 nm (El%Cm 1000),
240 nm (shoulder), 265 nm (shoulder) and 320 nm (El~Cm 156)
in a solution thereof in N/10 HCl-methanol (1:9) and by
absorption maxima at 228 nm (El%Cm 800), 254 nm (El~Cm 725),
291 nm (El~Cm 456) and 324 nm (El~Cm 466) in a solution thereof
in N/10 NaOH-methanol (1:9); giving an Rf value of 0.50 in
thin layer chromatography on silica gel with chloroform-
methanol (10:1 by volume) as the developing solvent.
This invention embraces neothramycin A and neo-
thramycin B, either alone or in a mixture of them, which may
be present in a dilute solution, as a crude concentrate, as
a crude solid, as a purified solid, as the free acid form
and in the form of a salt thereof with a metal or an organic
amine. Neothramycin A has been obtained as a colorless
powder which has no definite melting point, melts gradually
near 105C. and decomposes at 132-147C. with foaming and
which exhibits a specific optical rotation ~O~] 26 = +272
(C 0.52, dioxane). From the results of elemental analysis
and the determination of molecular weight, it is very probable
that neothramycin A has an empirical formula C13Hl4N2O4-1/2H2O.
This formula has been confirmed by high-resolution mass-
spectrometry (Found: m/e 262. 0934, calculated molecular
weight for C13H14N2O4 262 . 0952) . The ultraviolet absorption
spectrum of neothramycin A in an alkaline solution exhibits
a shi't towards the longer wave length. As shown in
3 0 Table 1, NMR spectrum of neothramycin A shows the presence
~,.,
1056747
of 14 protons. Neothramycin B is very similar in its
properties to neothramycin A and has been obtained as a
colorless po~Jder which has no definite melting point, commer.ces
to decompose at 144C. with foaming and completely melts at
151C. and which exhibits a specific optical rotation
[~]D6 = +314 (C 0.48, dioxane). Neothramycin B has the
empirical formula: C13H14N204-1/2H2O. This formula has
been confirmed by high-resolution mass-spectrometry (Found:
m/e 262.0939, calculated molecular weight for C13H14N2O4,
262.0952). The ultraviolet absorption spectrum of neothramycin
B in an alkaline solution exhibits a shift towards the longer
wave length. As shown in Table 1, N~R spectrum of neothramycin
B shows the presence of 14 protons, similarly to neothramycin A.
Neothramycin A and B are stable for a long period of time
when stored in the form of a solid powder thereof in a cold
and dark place.
But, neothramycin A and B are unstable in 50%
aqueous ethanol of pH 2.5 and the activities are reduced to
25% and 2Z~, respectively, at room temperature for 16 hours.
In 50~ aqueous ethanol of pH 6.5 or pH 8.0 at room temperature
for 16 hours, 80-90% activity of neothramycin A and 70-80~
activity of neothramycin B remain, but an equilibrium con-
version of neothramycin A to B or B to A is shown by thin-
layer chromatographic analysis.
From these data, neothramycins A and B are isomers
which are convertible into each other and belong to the
anthramycin group of antibiotics possessing a benzodiazepine
structure. They are distinguished from anthramycin, dextro-
chrysin and sibiromycin by their W spectra. The W spectra
of tomamycin and neothramycins are very simllar but they
-- 6 --
A~ jJ
~056747
TABLE 1
PMR Chemical Shifts of Neothramycins
Proton Neothramycin A ~eothramycin B
CH2x2 1.7-2.5 1.7-2.5
OCH3
CH 3.80 m 3.78 m
arom. OCH3 3.90 s 3.88 s
OH 5.00 d 5.10 d
CH 5.69 dd 5.7a m
arom. H 6.70 s 6.69 s
arom. H 7.43 s 7.40 s
CH 7.62 d 7.70 d
phenol OH 8.00 s 7.98 s
Chemical shifts, ~(ppm) were measured in deuterodioxane
using TMS as the internal reference.
1056747
are different in their molecular fo:rmulae and other
spectra.
By struc~ural studies, the following structures
have been submitted by the inventors for neothramycin A
(R = OH, R = H) and neothramycin B (Rl = H, R2 = OH).
CH30 ~ ~ R2
In another aspect of this invention there is
provided the process for producing the antitumor antibiotic
neothramycin selected from the group consisting of neothramycin
A, neothramycin B and mixtures thereof, said neothramycin A
having the formula:
HO ~ N =
CH30 ~ ~ N ~
O HO H
and said neothramycin B having the formula:
CH30 ~ o~= ~ OH
which comprises culturing a neothramycin-producing strain of
Streptomyces having the identifying characteristics of
A.T.C.C. 31123 under submerged aerobic conditions in a
nutrient medium containing a carbon source and a nitrogenous
- 8 -
~'
1056747
nutrient until a substantial amount of neothramycin is produced
by said organism in said nutrient medium, and recovering the
neothramycin from the nutrient medium when the pharmaceutically
acceptable salt of the neothramycin is desired, the process
further comprises a step of forming said salt by methods known
per se.
~eferring to the attached drawings: -
Figure 1 shows a curve of the infrared absorption
spectrum of a sample of neothramycin A pelleted in potassium
bromide.
Figure 2 shows a curve of the infrared absorption
spectrum of a sample of neothramycin B pelleted in potassium
bromide.
Figure 3 shows curves of the ultraviolet absorption
spectrum of a sample of neothramycin A dissolved in 10%
water-methanol, in N/10 NaOH-methanol (1:9) and in N/10
HCl-methanol ~1:9), respectively.
Figure 4 shows curves of the ultraviolet absorption
spectrum of a sample of neothramycin B dissolved in 10%
water-methanol, in N/10 NaOH-methanol (1:9) and in N/10
HCl-methanol (1:9), respectively.
The neothramycin A and neothramycin B of this
invention have low antibacterial and antifungal activity as
will be clear from the antibacterial spectra of these sub-
stances shown in Table 2 below. The minimum inhibitory
concentrations (mcg./ml.) of neothramycin A and neothramycin B
to various bacteria have been determined on nutrient agar
plates which were incubated at a temperature of 37C. for
17 hours. The minimum inhibitory concentrations to various
fungl have been determined on nutrient agar plates containing
1% gluco~e after lncubation at 27C. for 40 hours.
_ g _
,1~
1056747
Table 2
Minimum Inhibitory Concentratlons
(Mc~./ml.)
L3a~liam9 Neothramycin A Neothramycin B
Staphylococcus aureus Smith50 100
Staphylococcus aureus 209P~ 100 ~ 109
Klebslella pneumoniae PCI 602 50 100
Escherichla coli ~IHJ 100 100
Escherichia coli K-12 100 100
Pseudomonas aeruginosa No. 12 ~ lO0 > 100
Baclllus subtilis PCI 219100 ~100
E~cherichla coli W677 50 100
~scherichia coli JR66/W677lO0 > lO0
Aeromonas salmonecida ATCC 14174 25 5
Vibrlo angulllarum NCBM 6 50 100
Saccharomyces cerevisiae 50 ~ 100
Candlda albicans 31lL7 > 100 ~ 100
Aspergillus niger 100 ~ lO0
Piricularia or~zae 50 > 109
Xanthomonas cltrl ~ 100 > 100
Xanthomonas oryzae 50 100
-- 10 --
~ ,~/~,, .
1056747
As stated hereinbefore, neothramycin A and neo-
thramycin B of this invention have a high inhibitory
activity to the growth of leukemia cell3 and are
expected to be useful a~ an agent for treating
therapeutically a livlng anlmal affected by
leukemla, Chemotherapeutlc effects of neo-
thramycin A and B a~alnst leukemia L-1210 ln mlce
were inve~tigated ln the followlng manner, Leukemla
L-1210 cells (105 cells/mouse) were lnJected lntra-
perltoneally ln mice of CDF 1 straln welghing 19-22 g.
For the treatment of the leukemla 80 lnfected,
admlni~tratlon of neothramycin A and B were
¢ommenced lmmediately after the tumor inoculatlon.
The leukemic mlce were used ln group~ each of four
mlce for each dose, When 300, 150, 75, 37,5 and
18.7 mcg,/mouse/day of neothramycin A and B
were dosed by lntraperltoneal lnJection once dally
for 10 days, the hlghly favorable effect~ on the
~urvlval ratlo (%) were observed as wlll be clear
from the re~ults shown ln Table 3 below.
1056747
lable 3
Avera~e of Su~vlval Ratio
Do~a~e ~g~mQuge/day) Neothramycin A Neothram~cin B
300 death death
(toxlc dose) (toxic dose~
150 200 192
167 154
37.5 154 128
18.7 122 103
me Survival Ratio (%~ 1~ calculated by dlvldlng the
number o~ day~ of ~urvival of the treated animals
(e.g, 10) by the number of days o~ survlval o~ ~he
control animal3 (e,g. 8) and multlplying by 100,
e.g. ~ x 100 - 125. Ratio3 greater than 125 are
generallg con~idered ~ignlflcant.
The neothramycin A and B of this invention
are of a low toxlclty to anlmal and man, a~ ~hown
by the fact that the neothramycin A
!, ~ ~, 12
, ~ .
1056747
and -B exhibit LD50 values of 20 - 30 mg./kg. and
20 - 30 mg./kg., respectively, in mice, when a
solution of 0.25-0.5~ by weight of neothramycin A
or B in 10% dimethylsulfoxlde-water is injected
intraperitoneally in mice for the purpose of esti-
mating the acute toxicitv of these substances.
~ cccrding to a second aspect of this inven-
tion, there is provided a process for the production
of neothramycin A and neothramycin B, which compxises
cultivating a neothramycin-producing strain of the
genus Streptomyces under aerobic conditions in a
suitable culture medium therefor containing assimilable
carbon and nitrogen sources for a period of time
sufficient to produce and accumulate neothramycin A
and neothramycin B in the culture medium, and recovering
a mixture of neothramycin A and neothramycin B from
the culture, and subsequently, if reouired, separating
the recovered mixture into neothramycin A and neothramycin
B in their isolated forms. For the production of
neothramycin according to the process of this invention,
a strain of the genus Streptomycas may be used as lon~
as this strain produces neothramycin. ~ suitable
example of the strain which may be employed in this
invention for the production of neothramycin is the
above-mentioned MC916-C4 strain of Streptomyces.
1056747
Thl8 MC916-C4 strain was deposited on February 2, 1974
in a Japanese authorized deposltory "Fermentatlon
Research Institute, Agency of Industrial Sclence and
Technology", Inage, Chiba-City, Japan, under deposit
number FERM-P 2452. m is MC916-C4 strain was al~o
deposited in the Amerlcan Type Culture Collection,
Washington, D. C , U.S.A. under A.T.C.C. number ~1123.
Cultural and taxonomic characteristics o~ the
MC916-C4 strain are described below.
1. Microsco~ical mo~hQlQæ~
MC916-C4 strain has branched substrate mycelia
~rom which aerial hyphae develops in the form of hook
or open spirals. No whorl-branching ls observed,
Matured spore chains usually bear more than lO conidal
spores. Spores measure about o.6-o.8 by 1.0-1.2 mlcrons
in size and have a smooth surface.
2. Characteristics of the ~rowth on various culture media
m e designatlon of colors ln brackets [ ] mentioned
below follows the color standard given in the "Color
Harmony Manual" published by Container Corporation of
America.
(1) On sucrose-nitrate agar (incubated at 27 C.):
Pale yellow to reddish yellow [~ pc, amber] colored
growth bears thin aerlal hyphae o~ llght brownlsh gray
to pale gray color. Soluble plgment is faintly tinged
wlth yellow.
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~, ,
~05674~
(2) On glucose-asparagine agar (incubated at 27 C.):
Dull yellow orange ~nc, Amber to 4pe, Orange Rust]
colored growth develops aerial hyphae o~ light gray to
light brownish gray color [2ih, Dk covert Gray].
Soluble pigment is faintly tinged wlth yellow.
(3) On glycerol-asparagine agar [ISP No. 5 medium,
lncubated at 27 C.): Dark yellow orange to yellowish
brown ~pi, Golden Brown3 colored growth develops
aerial hyphae of brownish gray [~lh, Beige Gray] to
gray ~5ih, Shadow Gray] color. Soluble pigment with
yellowlsh tinge to yellowish brown tinge ls produced.
(4) On inorganic salt-starch agar (ISP No. 4
medium, incubated at 27 C.): Pale yellowish brown to
yellowish brown [3pl, Golden Brown] colored growth
develops aerial hyphae of llght brownish gray [3fe,
Silver Gray] color. Soluble plgment is tinged with
bro~tn. The reverse side of the growth is dark yellowish
brown in color.
(5) On tyroslne agar (ISP No. 7 medlum, incubated
at 27 C.): Dark yellow to yellowlsh brown [4pg, Dark
Luggage Tan] colored growth bears aerial hyphae of light
brownish gray. Soluble pigment is tinged wlth dark
yellow to yellowi~h brown.
(6) On nutrlent agar (incubated at 27 C.):
The growth is colored pale yellowish brown to pale brown
without developing aerial hyphae. Soluble plgment is
falntly tlnged with brown.
- 15 -
., ",
f ",
1~)56747
(7) On yeast extract-malt extract agar (ISP No. 2
medium, lncubated at 27 C.): Yellowlsh brown ~4pg.,
Dk Luggage Tan] to yellow orange [4pe, Orange Rust]
colored growth develops aerial hyphae of light gray
[2fe, Covert Gray to llght brownl~h gray [21h, Dk
Covert Gray color. Soluble plgment of yellowish
brown to brown color ls produced. The reverse slde
of the growth ls colored dark yellowlsh brown.
(8) On oatmeal agar (ISP No. ~ medlum, lncubated
at 27 C.): Reddlsh yellow to dark yellow orange
[4pe, Orange Ru3t] colored growth wlth aerial hyphae
of llght gray [5fe, Ashes] to brownlsh gray [31~,
Beige Gray] color, Soluble pigment ls tinged wlth
yellow.
(9) On glycerol-nltrate agar (incubated at 27 C,):
Pale yellow to reddlsh yellow [3pc, Amber] colored
growth bears ~lightly developed aerlal hyphae of
~ brownish white to light brownlsh gray color. Soluble
pigment is falntly tinged wlth yellow.
(10) On starch agar (incubated at 27 C.): me
growth 1~ colored dull yellow to yellowish brown
[2pi, Mustard Brown] without developing aerial hyphae
or rarely with developing aerial hyphae of white.
Soluble plgmsnt is falntly tinged wlth brown~
(11) On calclum-malate agar (incubated at 27 C,):
The growth ls colored pale yellow to pale ollve with-
out developlng aerial hyphae or wlth sllghtly developing
aerlal hyphae of whlte~ Soluble plgment is faintly
tinged with yellow.
.~,
~05674~
(12) On cellulose (incubated at 27 C,): Colorless
growth without aerial hyphae. No soluble pigment is
produced.
(13) On gelatin stab: On plaln gelatln medium
(incubated at 20 C.), the growth i~ colorles~ to dull
yellow colored wlthout developing aerial hyphae, and
wlth produclng soluble pigment of ~alntly yellow tlnge.
On glucose-peptone-gelatln medium (incubated at 27 C.),
the growth is pale yellow to dull yellow in color.
Aerial hyphae are not developed lnitially but ones of
grayl3h white color are produced later. No production
of ~oluble pigment is ob~erved.
(14) On skimmed mllk (incubated at 37 C.):
The growth is colored pale yellow to pale orange without
de~eloping aerial hyphae. Soluble pigment is very
faintly tinged with orange.
. Phvsioloical ~ro~erties
tl) Temperature for growth
Growth on gluco~e asparagine agar was examined at
20 C., 24 C,, 27 C., 30 C., 37C,, and 50 C. me
MC916-C~ strain grew at all temperaturea tested,
except at 50 C. Optimum temperature for good growth
was ob~erved to be ln the vicinity of 30 C.
(2) Liquefaction of gelatin
Plain gelatln (15%) medium ~tarted to liquefy from
the 5th day of lncubation at 20 C. me degree of
lique~action wa~ medium. me ~elatin (15%) in gluco~e-
peptone-gelatin medium started to liquefy from the 2nd
day of incubation when incubated at 27 C., and the
17 -
1056747
grade of liquefactlon was then medium to strong.
(3) Hydrolysis of starch
Starch in inorganic salts-starch-agar medium
and ln tarch-agar medium was hydrolyzed starting
from the 5th day of incubation when lncubated at
27 C. The grade of hydrolysis was medium to strong.
(4) Coagulation and peptonization of sklmmed
milX
When incubated at 37 C., the coagulatlon of
sklmmed milk started at the 4th day of incubation and
ths peptonization was observed at the 5th day of
lncubation after the coagulation was complete. The
grades of coagulatlon and peptonization were medium
to strong.
(5) Formation of melanoid pigment
No plgmentatlon was observed neither on trypton-
yeast extract broth (ISP No. 1 medium), nor on peptone-
yeast extract iron agar (ISP No. 6 medium), nor on
tyroslne agar (ISP No. 7 medlum), when lncubated at
27 C.
(6) Utllizatlon of carbon sources for growth
Utlllzatlon o~ the followlng carbohydrates was
tested ln Prldham-Gottlieb agar medium (ISP No. 9
medium) as lncubated at 27 C.
Glucose and L-rhamnose were utilized for growth.
L-Arablnose, D-fructose, sucrose, lnositol and D-
mannltol were not utllized. Utilizatlon o~ D-xylose
was doubtful. Rafflnose was sometlmes utilized but
- 18 -
,~
~f ~ ,
~OS6747
not utilized other tlmes.
(7) Liquefaction of calcium malate
Calclum malate ln calcium malate-agar medium was
liquefied around the growth starting at the 9th day of
incubation, when lncubated at 27 C. The grade of
llquefaction was medium to strong.
(8) Reduction of nitrate
Reduction of nitrate was observed ln aqueous
peptone solution containing 1.0% sodium nitrate
(ISP No. 8 medlum), when incubated at 27 C.
Summarizing the above-mentioned characteristics
of the MC916-C4 strain, it is noted that this strain
belongs to the genus StrePtomvce~ and that the aerial
hyphae form open spirals but does not develop whorl.
me surface of spore is smooth under microscopic
observation. On various media, the ~rowth has a color
of yellowish orange to yellowish brown with developing
aerial hyphae of light brownish gray to brownish gray
color. Soluble pigment ls tinged with yellow to brown
or with yellowish brown. No melanoid pigment is
produced, Proteolysis and starch hydrol-Jsis are of
medium to strong grade.
On the basis of the above-mentioned properties,
the MC916-C4 strain i~ compared to kno~ analogous
~pecle3 of Stre~tomvces with reference to descrlptions
of International Streptomyces Pro~ect (ISP). It is
found that the MC916-C4 strain resembles StrePtom~ces
naraen~is (see "Internatlonal Journal of Systematic
A~" - 19 -
1056747
Bacteriology" Vol. 22, page 32~ (19723. However,
it is noted that the MC916-C4 straln is dl~ferent
~rom Stre~tomvces naraensis ISP 5~o8 strain ln
respect to thelr utillzatlon of carbon sources.
S. naraensls produces cycloheximide, simllarly
to the MC916-C4 straln. Furthermore, among cyclo-
hexlmlde-produclng strains, it is found that stralns
of Group C whlch are analogous to Stre~tom~ces
~riseolus as reported ln an article by T. Furumai
et al. titled "On cycloheximlde-produclng micro-
organlsma" [see the "Journal of Antibiotics" Ser.
B., Vol. 17, No. 4, page 181 (1964)] are very
~imilar to the MC916-C4 ~traln,
me MC916-C4 straln ls well coincident wlth
the above Group C stralns ln many re~pects, though
the MC916-C4 straln has not been tested as to whether
it has the properties of hemolysls, liquefactlon of
serum and utilizatlon of galactose and lactose which
were shown by the Group C straln~. However, those
Group C stralns are not available at present, as
they are already dead. In this sltuatlon, comparlson
of the MC916-C4 straln ls now made wlth Stre~tomvces
sp, IFO 3300 whlch ls known to produce fermlcidln,
an antlblotlc analogous to cycloheximlde, and whlch
ls reported in the above article by T. Furumai et al.
to be well colncldent wlth sald Group C strains, The
result~ of comparlson are sho~m ir. Table 4 below, wlth
reference to the descrlptlons of the "Journal of
Antlblotlcs".
- 20 -
~'~ ''
1056747
O ~ S ~h
n5
~ P' rR h t ~m I I I ~ ~
~1 9 F: ~ S ~
~:
m C~ ~8 o
~ m
m mo ~ ~
a o ~ a~ ~ o
^ s ~ ~o~ *l *l * ~ I
m h ~ o ~ a~o
~S ~ ~ o ~
O u~ 1 cq ~c~ ~ ~ o
S-.l S
mo~ m ,13 o~ ~ ~
~o ~ ~:o ~ 3 ~ o
~~ m b~ 0.4 m ,~
o ~i ~ ~m~l ~
~1o o ~o o ~ ~ ~ S
'~ ~ ~H h ~ I I I ~ +
01:4H H ~ tU ~ O ~1
0 ~ ,c ~ ~ o Q~ 3C 3 b~
L h ~ 1~ I O
D. ~ ~ ~ ~ O t~ ~ O
G~ m~ 0 ~ ~ ~ ~ ~ Z o
S ~ 0 0~
~ ~OS
:~ S~ h O o ~ ~: S
V ~ O ~ ~1 ~
~O ~ ~ o~ ~ o~ o
H ~ O S h ,1 h 3 ~ ~'~ I I I I +
~ ~ ~ O b~a~ S ~ ~I O 'I
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-- 21 --
~05674 ~?
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-- 22 --
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~056747
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z
105674~
As will be seen from the above Table, the MC916-C4
strain is coincident with the Group C strain descri~ed in
the above-mentioned literature but is differentiated from
Streptomyces sp. IFO 3300 strain in respect to the coagulation
and peptonization of milk. Furthermore, the MC916-C4 strain
is different from Streptomyces griseolus [see the "International
Journal of Systematic sacteriology" Vol. 18, page 12~ (1968)]
which has been reported to resemble said IFO 3300 strain, in
that S. griseolus does not form open spirals in thè aerial
hyphae thereof and is somewhat different from the MC916-C4
strain in respect to the utilization of carbon sources. Further
comparisons of the MC916-C4 strain with Streptomyces sp.
IFO 3300, StrePtomyces griseolus ISP 5067 and Streptomyces
-
naraensis ISP 5508 are carried out. It has been found that the
MC916-C4 strain is related to Streptomyces sp. IFO 3300 and
Streptomyces naraensis ISP 5508 and most to the former strain.
The IFO 3300 strain is somewhat different from the MC916-C4
strain having a tinge of orange in the color of growth. The
MC916-C4 strain is clearly dlstinguished from said ISP 5508
strain in respect to the reduction of nitrate and from said
ISP 5067 strain in respect to the formation of spirals, utiliza-
tion of carbon sources and reduction of nitrate.
Mutation of actinomycetes occurs frequently in
either artificial or spontaneous conditions. Accordingly,
this invention includes the use of the MC916-C4 strain as
well as it mutants. In other words, this invention includes
the use of all strains of the genus Streptomyces which
produce neothramycin.
The neothramycin can be obtained by aerobic cultiva-
tion of spores or mycelia of a neothramycin-producing strain
- 24 -
.r -
105679L7
of the genus Streptomyces such as Strpetomyces sp. MC916-C4
strain (identified as A.T.C.C. 31123). In carrying out the
process of the second aspect of this invention, an amount of
spores or mycelia of a neothramycin-producing strain is
inoculated to a suitable culture medium therefor comprising
nutrient sources and is then incubated under aerobic conditions
so that there is obtained a culture broth containing neothramycin.
Generally, constituents of culture media commonly employed for
the cultivation of ordinary actinomycetes can be used for the
purpose of this invention. For instance, commercially avail-
able soybean meal, peanut powder, cotton seed powder, dried
yeast, peptone, meat extract, casein, corn steep liquor,
N-Z amine, ammonium nitrate, ammonium sulfate and the like
may be useful as the nitrogen carbon sources. Commercially
available carbohydrates such as glucose, starch, glycerol,
maltose, dextrin, saccharose, lactose, molasses and the like
as well as fat or oil are useful as the carbon source. In
addition, sodium chloride, calcium carbonate, magnesium
sulfate, manganese chloride, sodium phosphate or other inorganic
salts can be employed for the salt-additive in the culture
medium. Various heavy metal salts may also be added in trace
quantities, if required. Any of the nutrient materials
which are known for the cultivation of actinomycetes may
be employed in the process of this invention, as long as
it is assimilable by the neothramycin-producing strain
for the production of neothramycin.
* Trademark.
- 25 -
105~;747
For the production of neothramYcin on a
large scale, llquid cul~ivatlon ~ 5 preferred. Any
temperature at which the neothramycin-producing
strain 18 able to grow and produce the neothramycin
can be employed for the cultivation, but
a pre~erred cultlvation temperat.Lre i~ wlthln a
range of 2~ to 35 C. The cultlvatlon 18 contlnued
for a perlod of t~me ~u~flclent to produce and
accumulate a sufficient amount of neothramycin
A and B in the culture medium. For instance,
a culture medlum comprislng 2% glucose, 2% glycerol,
1.2% soybean meal, 1.0% cotton seed flour, 0.32~
calclum carbonate, 0.5% ~odlu~ chlorlde and 0.0005%
manganese chlorlde tetrahydrate was prepared and
sterilized at pH 6.8. -ml8 medlum was then lnoculated
with spores or mycella harvested from a slant culture
o~ the MC916-C4 strain. When it was ~hake-cultlvated
aeroblcally at 28 C., the productlon and accumulation
of neothramycin in the culture medium r.~ached
a maxlmum at the end o~ lncubatlon ~or 3 to 5 days,
Assay of neothramycin can be made using
Sta~hvlococcus aure.~s or Escherlchla coli as the
test organlsm accordlng to a 3tandard cup-plate method
whlch has usually been employed for the assay of
known antiblotics. Pure neothramvcin A which
was obtalned from the Example ~, descrlbed later, Or
thl~ inventlon may be used a8 an authentl¢ sample
whlch exhibit~ a potency of 1000 unlts per mg. In
case the other antlbiotic 3ubstances such as cyclo-
- 26 -
1056747
hexlmlde are ~lmultaneou~ly produced ~n the culture
broth of the MC916-C4 ~traln in additlon to the
~eothramycin, the culture broth may be washed
wlth ethyl acetate or other 3ultable organlc 301-
vent to remove such other antlblotic ~ub~tances
by extractlon. The remalning aqueou~ pha~e may
tben be lmployed for the a~say of the contents
of neothramycin A and B according to the
aforeaald ~tandard cup-plate method.
For the recovery o~ the neothramycin from
the culture medlum, the culture broth of the neo-
thramycin-producing ~train may elther be treated
wlth a sultable organlc solvent such as n-butanol
to provlde an extract of the neothramycin in
sald solvent or may be treated with a suitable
ad~orbent such a~ active carbon to make the neo-
thramycin adsorbed by the adsorben$. Distrlbuti4n
of the neothramycin A or neothramycin B
between n-butanol and water was examined, and lt is
found that the partition coefflclent of the neo-
thramycin in n-butanol/water ls greater than 5 at
a pH value of 2 to 7. Accordingly, the neo-
thramycin can be extracted with n-butanol from the
aqueou~ culture broth whlch ha~ been ad~u3ted to
a pH value of 2 to 7 and pre~erably of about 6.
The neothramycin is substantially insoluble
in and hence 18 practically not extractable with
ethyl acetate or chloroform from the liquld portion
- 27 -
; r;
1056747
of the culture broth. If required9 there~ore, lt 18
posslble to treat the culture broth wlth ethyl aoetate
or chloro~orm for extractlon in order to remove the
soluble impuritles ~rom the culture broth. To
separate the neothramycin from the culture broth
lt ls pre~erred that the culture broth ls treated
wlth actlve carbon a~ the adsorbent. The neo-
thramycin whlch has been ad~orbed by active carbon
can be eluted thererrom by means o~ a mixture o~
methanol and water, a m~xture of propanol and water
or a mlx$ure o~ acetone and water, etc. The efflclency
of the elution may be lmproYed when the elutlon ls
done under weakly alkallne condltions. Purlfication
o~ the neothramycin can be made uslng the above-
mentloned extractlon method and adsorption-elutlon
method ln a ~ultable comblnatlon of them or ln a
repeated manner. Further puriflcatlon may be achleved
b~ a u~ual column chromatography on Sephadex IH-20
(a commerclal product sold by Pharm~c~a Co., Sweden)
or slllca gel. The known antlblotlc cycloheximlde
whlch may fr~quently be co-existent ln the culture
broth of the MC916-C4 strain can readll~ be sepQrated
from the neothramycin of this invention by
extracting with eth~l acetate or b~ chromatogra~hy
on Sephadex I~-20
To lsolate the neothramycin A from the neothramycin ~,
a mixture of the neothramycin A and
*Trade Mark
- 28 -
~ ~i
1056747
neothramycin ~ may be subjected to a column
chromatography on ~llica gel wlth chloroform-
methanol (30:1 by volume) as the developing ~olvent.
~he isolated neothramycin A or the isolated
neothramycin B can be purified by column
chromatography on 3111ca gel using sultable mixed
organlc solvents as the de~eloplng sol~ent~
The recovery Or the neothramycin A and neo-
thramycin B may typically be carried out in the
following way: ~he culture broth contalnlng the
neothramycin ls at first rlltered or centrlfuged
to remove the ~olld matters together with the
mycella. The broth flltrate 18 then treated
wlth actlve carbon to adsorb ~he neothramycin
there~rom. The actlve carbon carrying the ad~orbed
neothramycin is eluted with 50% acetone-water
(a mlxture of acetone and water at a ratlo of 1:1
by volume) at pH 8Ø ~he eluate 18 collected ln
rractlon~ and the actlve fractlon~ are combined
together and concentrated to dr~ness under reduced
pre~ure at a temperature Or up to 40 C. or
otherwl~e ~reeze-drled to glve a crude powder.
m 1~ crude powder 18 extracted with aqueous
ethanol 80 that a greater part Or the actlve
component3 18 separated in the resultlng extract.
Thls extract i8 concentrated to drynes~ under
reduced pre~ure at a temperature Or up to 40 C.
- 29 -
~,,
1056747
or otherwise freeze-dried to give a second crude powder.
A solut~on of this crude powder in methanol 1~
passed through a column of Sephadex IH-20 (a product
of Pharmacla Co., Sweden) whlch is subsequen~ly developed
wlth methanol. Durlng thl~ chromatographlc proces~,
the posslbly co-existing cyclohexlmlde 13 eluted
in ~uch ~ractlons runnlng out ln the fir~t-half
phase of the process, whereas the mlxture of the
neothramycin A and B is eluted in such fractions
runnlng out ln the later-half phase of the proce~.
The actlve ~ractions containing the neothramycin
A and B are combined together and then con-
centrated to dryness under reduced pres~ure at a
temperature o~ up to 40 C" to a~ford a crude
powder. Ihls powder i9 taken into a small volume
of methanol and the methanollc solutlon is unl~ormly
admixed wlth an amount o~ neutral silica gel. The
admixture was drled by evaporatlon and then placed
on the top of a column of a further amount of said
neutral slli¢a gel which has been ~mpregnated wlth
a mixture of chloroform and ethanol (30:1 by volume).
~he sillca gel column i~ sub~equently developed with
the chloroform-ethanol (30:1 by volume). During thls
chromatograph~ proce~s, the neothramycin A is
eluted ln the actlve fractlons runnlng ln the first-
half pha~e o~ the proce~s, whlle the neothramycin B
18 eluted in the actlve fractlons runnlng out ln the
later-half phase of the process. The actlve fractlons
- 30 -
~' .
1056747
contalnlng the neothramycin A and the active
~ractions contalnlng the neothramycin B are
concentrated to dryne3s under reduced pressure at
a temperature o~ up to 40 C., respectively, to
glve a crude powder of the neothramycin A and a
crude powder o~ the neothramycin ~.
The crude powder of the MC916-A substance 80
obtained 18 taken lnto an appropriate amount of
chlorororm and the solutlon 18 passed through a
column of a neutral slllca gel whlch has been
lmpregnated wlth chloroform. ffl 19 slllca gel
column ls washed wlth chloroform and then developed
at 5 C. wlth chloro~ormrethanol (60:1 by volume).
The eluate 1~ collected ln ~ractions, and the
desired actlve fractlons solely contalnlng the
neothramycin A are detected by referring to
test results of blologlcal assay and thln layer
chromatography o~ each rractlon. me deslred
actlve fractlons so chosen are comblned together
and concentrated to dryness under reduced pre~sure
at a temperature of up to 40 C. to glve the neothramycin
A as a colorless powder. This powder may
~urther be purl~led to a colorless powder of pure
neothramycin A by repeating the above-mentioned silica gel
chromatographlc proce~s or by dlssolvlng sald powder
lnto a sm~ll volume of chloro~orm, addlng ethyl
ether to the chloro~orm solutlon, fllterlng o~ and
dry~ng the resultlng precipltate. A colorless powder
- 31 -
,;., ~
, . .
~, . ...
~056747
of pure neothramycin B may be obtained from the
a~oresald crude powder o~ the neothramycln s by
purlfylng in the ~ame manner a8 for the neothramycin A.
It is preferred, however, that the
column chromatography on slllca gel 18 made u~lng
a mlxture of chloro~orm and ethanol (lOO:l by
volume) as the developlng solvent.
In view of the aforesaid propertles of the neo-
thramycin A and neothramycin B, it has been con-
flrmed that theqe substance~ are new antiblotic3
whlch are dlf~erentlated from any of the known
antlblotlc~. Accordlng to a khlrd aqpect o~ thls
lnventlonJ there 1~ pro~lded a method for thera-
peutlcally treatlng a llvlng anlmal, lncluding
man, a~ected by leukemla, whlch comprl~es admln-
lstering the neothramycin A and/or neothramycin B
~ubstance to sa$d anlmal ln a do~age ~uf~lclent to
reduce the affection by leukemia. Accordlng to a
~ourth a~pect of this ln~entl~ , there 1~ ~urther
provlded a pharmaceutlcal compo~ition comp~l~tng
the neothramycin A and/or the neothramycin B
ln an amount ~uffl¢ient to reduce the affectlon by
leukemia ln vlvo, the neothramycin A and/or the
neothramycin B being in combina~ion with a
pharmaceutlcally acceptable carrler. It will be
appreciated that the actual pre~erred amount~ of
the neothramycin uséd will vary according to
- 32 -
, ,
, .,, . ~,
1056747
the partlcular compound belng used, the partlcular
compositlon formulated, the mode o~ appllcatlon and
the partlcular sltus and organlsm belng treated.
Many factors that modlfy the actlon of the drug wlll
be taken lnto account by the skilled ln the art,
~or example, age, body weight, sex, dlet, time of
adminlstratlon, route of adminlstratlon, rate Or
excretlon, drug combinatlons, reactlon sen~ltlvlties
and severlty of the dlsease. Optlmal applicatlan
rate~ ~or a glven set of condltlons can be a~certalned
by the skllled ln the art uslng conventional dosage
determinatlon tests in view o~ the above g ldelines.
It ls belleved that uslng the precedlng descrlptlon
and wlthout ~urther elaboratlon, one skllled ln the
art can utll~ze the concept of thls inventlon to lts
~ullest extent. The ~ollowing preferred speciflc
embodiments are, therefore, to be construed as
merely illustratl~e and not llmltative of the re-
malnder of the dl3closure ln any way.
De~crl~tion of the Preferred Embodlmen~
- 33 -
1056747
am~le 1
A loopful quantl~g o~ Stre~tom~ce~ sp. MC916-C4
(ldentlfied as A.T.C.C. 31123) whlch wa~ incubated
in slant agar medlum ~a~ inoculated to a sterile
llquid culture medlum (pH 7.0~ 125 ml.) compri~ing
2.5~ malto~e, 0.75% peptone, 0.75% meat extract,
0.3% yeast extract, 0.3~ sodlum chlorlde and 0.1%
magneslum sulfate (7H20). The inoculated medlum
waa shake-cultured at 28 C, ~or 48 hours to give
a primary seed culture. This prlmary seed culture was
inoculated at an inoculum slze of 0.48% by volume
to 50l or a ~terllized liquld culture medium (pH
7.0) oontainlng 3,5% ~tarch ~yrup, 0,75% peptone,
0.75% meat extract, 0.3% yeast extract, 0.3% ~odlum
chloride and 0.1% magneslum sulfate (7H20) ln a
stainless steel fermentor of a capacity of 130R .
The lnoculated medlum was cultured at 28 C. for
24 hour~ under aeratlon and agltation to provide
a secondary seed culture. This secondar~ seed
culture wa~ lnoculated at an inoculum size of
2% by volume ( 6l) to a liquld culture medium
(pH 6.8, 300l) comprl31ng 2% glucose, 2% glycerol,
1.2% soybean meal, 1.0% cotton seed powder, 0.3$
calcium carbonate, 0.5% sodium chloride and 0.0005%
~anganese chloride (4H20) whlch had been sterlllzed
at 120 C. for 30 mlnute~. The cultivation was made
ak 28 C. for 92 hour~ under aeration and agltatlon
(250 r.p.m.) whlle the rate Or aeration was 150
~¦ mlnute for the flrst 24 hour~ and then lncreased
- 34 -
105674~.7
to 300 ~/minute ~or the ~ubsequent period o~ 24th
hour to 92 hour of the cultivation.
The resulting culture broth (pH 7.3, 300R,
potency 88 u./ml.) wa 8 admixed wlth 24 kg. o~ a
filter ald (diatomaceous earth commerclally
available under a trade mark HIFR0-SUPERCEL)
and the admixture was flltered by mean~ of a press-
~ilter to give 300Q of the broth ~iltrate. The
broth flltrate wa~ well admixed with 3 Kg. o~
active carbon at ambient temperature for 1 hour
under agitation, so that the antiblotics were
ad~orbed on the carbon.
The active carbon portion was collected b~
centrlfugation and then washed with 150 l Or
water. m e washed carbon was admixed with 70
of 50% acetone-water (pH 8.0) ~or 1 hour under
agitation, 80 that the antib$otlcs were extracted
into the ~olvent. This extractlon was conducted
twice and the extracts 80 obtalned were combined
together to a volume of 118~. The extract solution
was concentrated under reduced pressure at a
temperature Or up to ~0 C. and the concentrated
solutlon (2.6Q) was freeze-dried to give 565 g. of
a brown colored powder (potency, 35 u./mg.) which
contained the neothramycin A and B. This
brown colored powder wa~ extracted wlth 11.6~ o~ 80%
ethanol-water. The $nsoluble matters which had no
antlbacterlal act$vlty were removed by flltratlon,
10567~
to yleld 11.2Q o~ an ethanollc extract. Thls extract
was concentrated under reduced pre~sure at a temperature
o~ up to 40 C. to a volume o~ 800 ml.9 and the
concentrated solutlon was freeze-dried to a~ord 218.5
g. of a crude powder (potency, 53 u./mg.). Thi8 crude
powder was dlvlded into rlve equal part~, and each
part was dl~olved in 20 ml. of methanol. me
methanol~¢ solutlon wa~ passed through a column
(90 mm diameter) of 4~ o~ Sephadex LH-20, which was
subsequently developed with methanol. lhe eluate
was collected ln 200 ml. rractlon~, and lt was round
that cyclohexlmide wa~ eluted out ln the rractlon
Nos. 9 to 11 whlle a mixture Or the neothramycin A and B
~ubstances was eluted ln the rractlon No~. 12 to 14.
The fra¢tion Nos. 12-14 were combined together and
concentrated to drynes~ under reduced pres~ure at a
temperature Or up to 40 C. to give 47 g. Or a crude
powder (potency, 160 u./mg.) comprl~lng the mlxed
neothramycin A and B. Yield 28% (based on the
neothramycin content of the culture broth).
Exam~le 2
The crude powder (3~ g.) comprlsing the mlxed
neothramycin A and B obtained in Example 1 was
taken lnto a small volume Or methanol, and the
solutlon was unlformly mlxed wlth 60 g. of a neutral
slllca gel, followed by drylng under reduced pres~ure.
The drled mas~ 80 obtalned was placed on the top o~
- 36 -
A~
lOS6747
a column (60 mm diameter~ Or 660 g. Or sald neutral
sillca gel which had been lmpregnated with chlorororm
ethanol (30:1 by ~olume). Th13 3111ca gel column was
developed at 5 C. by passlng a ~low oP chloro~orm-
ethanol (30:1 by volume) through sald column. The
eluate was collected ln 130 ml. rractlons, and
lt was found that the neothramycin A was eluted
in the rractlon Nos. 2~-31 whlle the neothramycin B
was eluted ~n the fractlon Nos. ~5-49. The combined
actiYe fractlon Nos. 23-31 Wa3 concentrated to dr~nes~
under reduced pressure at a temperature of up to
40 C. to glve 1.47 g. of a yellowl~h crude powder of
the neothramycin A (potency, 520 u./mg.). Yield
14%. me ¢ombined rractlon Nos. 35-49 were concentrated
to dryness ln the ~ame mQnner to gl~e 1.0~ g. Or a
yellowi~h crude powder Or the neothramycin B
(potency, 450 u./mg.), Yleld 9%.
Exam~le ~
The yellowtsh crude powder o~ the neothramycin A
(1 g.) obtained in Example 2 was dissolved in
20 ml. of chlorororm, and the solution was pas~ed at
5 C. through a column (1~ mm dlameter) o~ 20 g, of
a neutral sillca gel of the same grade as employed in
Example 2 whlch had been lmpregnated wtth chloro~orm,
The column was washed wlth 400 ml. of chloroform and
subsequently developed wlth chloroform-ethanol (60:1
by volume). The eluate Wa8 collected ln 8 ml. fractlons,
- 37 -
"~, ~,.,; ~ , .
10567~'7
and it was round that the neothramycin A was
eluted in the ~ractlon Nos. 13-27. me combined
Practlon Nos. 13-27 was concentrated to dryne~3 under
reduced pressure at a temperature oP up to 40 C.,
glving ~20 mg. of a raintly yellow colored powder.
Thl~ powder was taken lnto a mlnlmum volume oP
chloroform and to thls solutlon was added ethyl
ether until the precipltate ~ormed was not longer
depo~ited. ~he preclpltate wa 8 removed by
filtration and drled, a~fording 183 mg. of a
colorles~ powder of pure neothramycin A (potency
1000 u./mg.). Yield 35%.
Exam~Dle 4
me yellowi~h crude powder o~ the neothramycin B (810 mg.)
obtained in Example 2 was taken into 16 ml. of chloro-
Porm and the ~olutlon was passed at 5 C. through a
column (1~ mm dlameter) o~ 16 g. of neutral slllca
gel oP the same grade as employed ln Example 2 whlch
had been lmpregnated with chlorororm. The column
was washed with ~20 ml. of chloroform and then
developed wlth chloroPorm-ethanol (100:1 by volume).
me eluate was collected in 6.4 ml. Practlons, and
it was round that the neothramycin B was eluted
in the fractlon Nos. 34-70, The combined fraction
Nos. ~4-70 was concentrated to drynes~ under reduced
pre3~ure at a temperature of up to 40 C, to glve 160
mg. Or a faintly ~ellow colored powder, Thl~ powder
was taken lnto a mlnlmum volu~e oP chloroPorm, and to
- 38 -
105674~
thls ~olution was added ethyl ether untll the pre-
clpitate wa~ no longer deposlted. The preclpltate
was removed by rlltratlon and dried, a~ording 85
mg. of a colorles~ powder o~ pure neothramycin B
(potency, 620 u./mg.). Yleld 14.6%.
The Sephadex IH-20 used ln the precsding
example~ can be replaced by other slmilar gel-
*
flltratlon agent~, e.g. Sephadex G25 to G200,Sepharose ~B and 6B (Pharmacia Flne Chemlcals AB,
Uppsala, Sweden) and Blo-Gel Al.5m (Blo Rad Co.).
Prererred gel-~iltratlon agent~ include the
carboxymethyl substltuted cross-llnked dextran
gels descrlbed in ¢olumns ~ and 4 of U.S. patent
3,819,836.
The pharmaceutlcally acceptable salt3 o~ the
substances of the present lnventlon include nontoxic
metallic salts such a~ sodium, pota~ium, calcium and
aluminum, the ammonium salt and substituted ammonlum
salts, e.g. salts of such nontoxic amines as tri-
alkylamlnes includlng triethylamine, procaine, dl-
benzglamine, N-benzyl-beta-phenethylamlne, l-ephenamine,
N,N'-dlbenzylethylenediamine, dehydroabletylamlne,
N,N~-bls-dehydroabietylethylenedlamine, N-(lower)-
alkyl-plperldine, e.g. N-ethglplperidine, and other
amlnes whlch have been used to form ~alts wlth benzyl-
penlcillln
*Trade Mark~
A~ 39 _
