Note: Descriptions are shown in the official language in which they were submitted.
- ~0~4~3
1 This is a divisional application of patent application
serial number 291,230 filed on November 18, 1977.
This invention relates to a preventive and curative
treatment of tumors and a new derivative of ~-1,3-glucan usable
for the treatment.
For years there have been searches for substances which,
with parenteral or oral administration, will cause the inhibition
of tumor growth and the prolongation of the survival time of
tumor-bearing warm-blooded animals without serious side
effects.
It has now been found by the present inventors that a
water-insoluble, thermogelable ~-1,3-glucan produced by micro-
organisms belonging to the genus Alcaligenes and the genus
A~robacterium, partially hydrolyzed lower polymers thereof,
or carboxymethylated derivatives of said glucan or of said
lower polymers have a strong antitumor activity against the
growth of tumors such as sarcoma, carcinoma or leukemia in warm-
blooded animals without undesirable side effect.
The principal object of the present invention is,
therefore, to provide a treatment for warm-blooded animals affected
or may be affected by neoplasias with the ~-1,3-glucan, the
lower polymers, or their carboxymeth~lated derivatives. And
another o~ject is to provide an antitumor agent containing
the ~-1,3-glucan, the lower polymers, or the carboxymethylated
derivatives as active ingredients. A further object is to
provide novel carboxymethylated derivatives of either the
~-1,3-glucan or the lower polymers obtainable by partial
hydrolysis of the ~-1,3-glucan.
It is already known from Japanese Patent Publications
30 Nos. 32673/1973 and 32674/1973 and British Patent No.1,352,938
that certain strains of microorganisms belonging to the genus
'~
`' 109~453
1 Alcaligenes and the genus Agrobacterium produce a water-
insoluble, thermogelable ~-1,3-glucan (hereinafter referred
to briefly as TAK-N). However, no pharmaceutical usage of the
glucan has been known at all.
The extensive studies made by us on TAK-N, lower
polymers obtainable by partial hydrolysis thereof (hereinafter
referred to as TAK-D) and carboxymethylated derivatives of
TAK-N or of said lower polymers (hereinafter referred to as
CMTAK) led to the finding that these substances possess a
strong antitumor activity giving a markedly high tumor
inhibition ratio and a complete regression of tumors. The
finding was followed by further studies which have resulted
in the completion of this invention.
This invention is directed to a method of inhibiting
the growth of a tumor in a warm-blooded animal which comprises
administering to said animal to inhibit the growth of said
tumor a sufficient amount of a member of the group consisting
of a ~-1,3-glucan which is water-insoluble and thermogelable in
the presence of water at an average degree of polymerization of
170, a lower polymer obtainable upon partial hydrolysis of said
glucan and a carboxymethylated derivative of said glucan or
said lower polymer.
And another part of this invention is directed to
an antitumor agent containing said glucan, said low polymer
or said carboxymethylated derivative.
TAR-N which is employable in accordance with this
invention is such that, as described in detail in the afore-
mentioned patent literatures,it is produced by the cultivation
of certain microorganisms such as Agrobacterium radiobacter
(IF0 13127, ATCC6~661. Agrobacterium radiobacter U-l9(IF0 13126,
ATCC 21679, FERM P-1166) and Alcaligenes faecalis var. myxogenes
. ~
-- 2
A
. . .
. . .
. . .
.
.
-` 109~53
1 NTK-u (IFO-13140, ATCC 21680, FERM-P 1168). This ~-1,3-glucan
contains ~-1,3-bonds as its glycosidic linkages and is water-
insoluble, most species of which have average degrees of poly-
merization (hereinafter abbreviated as DP) not less than about
70 and a characteristic property such that they are gelable at
DP 170 when heated, for example at about 60C, in the presence
of water.
The DP of TAK-N is variable according to the method of
preparation. Thus, as determined by the method of Manners _ al
10Carbohydrate Research 17, 109 (1971), it is from about 70 to
1,000 and, in many cases, from 100 to 600. And, while these
variants of glucan species are generally thermogelable in the
presence of water, they lose gelability as their DP values are
lowered. For example, the glucan with DP of 113 is not gelable
but that with 170 is gelable. We have discovered that all of
TAK-N, TAK-D and CMTAK have a potent antitumor activity.
It is particularly surprising that even some species
of TAK-D which may not be called polysaccharides because o~
their low degree of polymerization retain and display such an
antitumor activity.
TAK-D is produced by partial hydrolysis of TAK-N. Among
the methods of hydrolysis available for this purpose are such
known methods as acid hydrolysis, alkaline hydrolysis and
enzymatic hydrolysis with ~-1,3-glucanase.
TAK-D can be separated from the reaction mixture by
various procedures that are commonly practised for the puri-
fication or fractionation of polysaccharides and oligosaccharides,
such as precipitation under acidic conditions, precipitation by
the addition of ethanol, and gel filtration. By such procedures,
various lower polymers each having the desired DP value can be
obtained separately.
.
109~4~3
1 Ther term T~K-D used throughout this specification means
any and all types of lower polymers which are obtainable by ,
partial hydrolysis of TAK-N.
As aforesaid, DP of TAK-N as obtained from cultures of
the aforementioned microorganisms are broadly dispersed. And
since the degree of polymerization of the ~-1,3-glucan produced
in the course of the fermentation process tends to decrease as
the cultivation time is prolonged, it is even possible to
produce TAK-N with DP lower than the above-mentioned range.
On the other hand, since TAK-D is produced by the hydro-
lysis of T~K-N, its DP is naturally lower than the starting
material TAK-N. But, generally speaking, TAK-N having degrees
of polymerization corresponding to those of a large majority
of TAK-D species can be produced by the cultivation of said micro-
organisms. And so long as TAK-N and TAK-D have the same DP, no
substantial difference in antitumor activity and thermogelability
has been found between them.
The manner of production of TAK-D from TAK-N will now
be described by way of the results of our experimental
production runs.
Experiment 1 (hydrolysis with sulfuric acid)
In 6 liters of 4 N-sulfuric acid was suspended 60 g of
TAK-N (DP : 540 as measured by the method of Manners et al.
Unless otherwise specified hereinafter, all DP values indicated
were measured by this method~. The hydrolysis reaction was
carried out at 60C. After 30 minutes and after one hour,
a 2 liter portion of the reaction mixture was taken. These
portions of the reaction mixture were designated Sl and S2,
respectively. The remainder of the reaction mixture was further
incubated at 6QC for an additional one hour to obtain another
sample S3. From Sl, S2 and S3, hydrolysis products were prepared
-- 4 --
, ~ :
-` 105~44~3
1 by the following procedure. First, each sample was centri-
fuged and the precipitate was collected. Each precipl-tate was
then washed with 1.6 liters of distilled water and centrifuged.
This procedure was repeated again and the precipitate was
suspended in 1.2 liters of distilled water. The suspension
was neutralized with an 8 N-solution of sodium hydroxide and
lyophilized. The above procedure provided 22 g, 20 g and 19 g
of powders, from Sl, S2 and S3, respectively. Then 7 g portions
of these powders were respectively suspended in 700 ml of
distilled water and the pH was adjusted to 12.5 with an 8 N
solution of sodium hydroxide to yield Sl', S2' and S3',
respectively.
Ethanol was added to each solution to give a final con-
centration of 60 % and, after the resulting precipitate was
removed by centrifugation, ethanol was further added to the
supernatant to give a concentration of 70 %. Then, the solution
was neutralized with dilute hydrochloric acid. The resulting
precipitate was collected by centrifugation, washed 4 times with
250 ml portions of distilled water and, then, lyophilized.
In this manner, 1.8 g and 1.5 g of white powders (S-I
and S-II~ were obtained from Sl' and S2', respectively. To
S3' was added a sufficient amount of ethanol to give a final
concentration of 70 % and the mixture was centrifuged. The
precipitate was resuspended in about 500 ml of distilled water
and, after the addition of ethanol to give a concentration of
70 %, the suspension was neutralized with a small amount of
hydrochloric acid. The resulting precipitate was collected by
centrifugation, washed 4 times with 250 ml portions of
distilled water and lyophilized, which provided 5.6 g of white
powder (S-III).
-- 5 --
~0944~;3
1 The physioehemical properties of S-I, S-II and S-III are
shown in Table l.
TABLE l
S-I S-II S-III
_*
DP 125 82 68
Elemental analysis(%)
C40.1340.65 40.94
H6.74 6.77 6.76
Purity(%)** 93.1 91.1 93.8
Glucose content(%)*** C0.1 ~0.1 ~0.1
[~]25 in 0.1 N NaOH 25.6 19.5 18.8
(C=1.16)(C=1.10) (C=0.82)
* Measured by the method of Manners et al
** Calculated from glucose content determined by the
phenol-sulfuric acid method
*** Measured by gas chromatography
Experiment 2 (hydrolysis with formie acid)
(I) In 150 ml of 85 % formie aeid was dissolved 6 g of TAK-N
(DP : 540) and the hydrolysis reaction was earried out at 88C
for 20 minutes. After eooling, the reaction mixture was con- -
centrated to dryness and the eoneentrate was suspended in water.
The suspension was brought to pH 12.5 with a 5 N-solution of
sodium hydroxide to obtain a elear solution. The solution was
brought to pH 7.0 with 5 N-hydroehlorie acid and the resulting
preeipitate was eolleeted by eentrifugation. The preeipitate
was washed well with distilled water and lyophilized. By the
above proeedure was obtained 5.4 g of white powder (F-I).
(II) Twelve grams of the same TAK-N as used in (I) was hydrolyzed
in 300 ml of 90 % formic acid at 95C for 20 minutes and the
reaction mixture was treated in essentially the same manner as
-` 10~453
1 in (I) to obtain 10.3 g of white powder (F-II).
(III) Twelve grams of the same TAK-N as used in (I) was
hydrolysed in 300 ml of 90 ~ formic acid at 95C for 40 minutes
and the reaction mixture was treated in essentially the same
manner as in (I) to obtain 5.4 g of white powder. The powder was
dissolved in 0.05 N-aqueous solution of sodium hydroxide to
give a final concentration of 1.0 % and ethanol was further added
to the solution to give a concentration of 57.5 %. The resulting
precipitate was collected by centrifugation, suspended in dis-
tilled water and neutralized with dilute hydrochloric acid. Thesuspension was centrifuged and the precipitate was washed well
with 70 ~ aqueous ethanol and lyophilized to obtain 2.2 g of white
powder (F-V).
To the supernatant obtained upon recovery of the above
ethanolic precipitate was added ethanol to give a concentration
of 70 % and the mixture was neutralized with hydrochloric acid.
The resulting precipitate was collected by centrifugation,
washed with 70 % aqueous ethanol and lyophilized. By the above
procedure was obtained 2.3 g of white powder ~F-III). The
supernatant remaining after recovery of the first precipitate
~5.4 g) from the hydrolysate mixture contained a substantial
amount of partial hydrolysates in a solubilized state. Therefore,
this supernatant solution was concentrated and centrifuged to
separate the generated precipitate. The precipitate was lyophilized
to recover 2.9 g of white powder (F-VII~. The resulting
supernatant solution was further fractionated by gel filtration
chromatography on a column of Sephadex* G-25 (solvent: 0.1 M
ammonium bicarbonate~ followed by lyophilization of combined
fractions. By this procedure was obtained 0.7 g of white powder
; 30 (F-VIII~-
*Trade Mark
1t)~44S3
1 (IV~ The same TAK-N (18 g) as that used in (I) was hydrolyzed
in 450 ml of 90 % formic acid at 95C for 40 minutes and, to
this reaction mixture, essentially the same fractionation pro-
cedure as in (III) was applied, such as concentration, pre-
cipitation with ethanol, gel filtration, etc. sy the above
procedure, four fractions of white powder (F-IV, 2.3 ~, F-VI 2.8 g,
etc.) were obtained.
The physiochemical properties of these powders - F-I,
F-II, F-IIX, F-IV, F-V, F-VI, F-VII and F-VIII - are shown in
Table 2. When six samples having lower DP (F-III, IV, V, VI,
VII, VIII) were each dissolved in a 0.02 N-solution of sodium
hydroxide and, chromatographed respectively on the same Sephadex****
G-200 column equilibrated with the same solution as above, they
were eluted in the order of decreasing DP (from F-III to F-VIII),
each sample giving a symmetrical peak, which indicated that each
sample had a normal distribution in regard to degree of
polymerization.
TABLE 2
DP Elemental Purity Glucose[a]D5
analysis(~) (%)** content in 0.1 N NaOH
F-I 299 40.73 6.46 92.3< 0.03 30.6(C=0.35)
F-II113 41.71 6.91 98.4< 0.03 22.3(C=0.57)
F-III50 41.98 6.77 93.5< 0.03 12.1(C=1.06)
F-IV 44 42.35 6.50 90.8< 0.03 10.9(C=2.24)
F-V 39 41.44 6.71 93.3< 0.03 4.0(C-1.62)
F-VI 24 42.62 6.99 92.5< 0.03 -1.5(C=1.07)
F-VII16 42.46 6.99 91.8< 0.03 0.4(C=1.06)
F-VIII7 39.84 6.37 94.1< 0.03 0.0(C=0.57)
*, ** and ***: see the footnote to Table 1.
****Trade ~ark - 8 -
, ~
--`` 109~4~3
1 We have also discovered that the carboxymethylation oE
TAK-N and TAK-D yields new derivatives (CMTAK) in which hydroxyl
groups of these glucans are more or less carboxymethylated.
Some aspects of this invention are predicated upon the
above finding. Thus, the invention is also directed to ~-l,3-
glucan derivatives of the following general formula and their
salts.
2 2 ~H2R
~ ~ H ~ 0 ~ ~ ` (I)
OR~ ~ ~ J ~1, OR
(wherein at least one of R's is -CH2COOH, with the remainder,
if any, being H; n is zero or an i~tegral number; or its salt.
C~qTAK can be obtained by carboxymethylating TAK-N or
TAK-D in a known manner, for example by reacting TAK-N or T~K-D
with monochloroacetic acid in the presence of alkali. In
addition to this method, any other conventional methods that are
used for the carboxymethylation of carbohydrates may be
- employed.
To recover CMTAK from the reaction mixture, it is
possible to employ conventional methods that are commonly used
for the purificatlon of carbohydrates, such as precipitation by
the addition of an organic solvent. In addition, such other
procedures as ion exchange chromatography, gel filtration, etc.
; may likewise be utilized according to the content of carboxymethyl
groups in, or the solubility in water of, CMTAK.
While the CMTAK thus obtained contains carboxymethyl
; 30 groups in its molecule, the content of carboxymethyl groups
varies widely depending upon the conditions of the carboxymethyla-
_ 9 ~
. . .
- 10944~3
I tion reaction. As determined by titration, the content of
carboxymethyl groups of the CMTAK obtained by a normal carboxy-
methylation reaction procedure is generally less than 3 carboxy-
methyl groups per glucose residue in the CMTAK molecule. CMTAK,
as it is suitable for the p~poses of this invention, includes
all substances which are obtainable by the carboxymethylation
of TAK-N or TAK-D and the molecules of which contain detectable
carboxymethyl groups, irrespective of the degree of carboxy-
methylation.
As will be apparent from the general formula (I) given
hereinbefore as its free acid, CMTAK can react with various
bases to form the corresponding salts, such as the sodium,
potassium, calcium, aluminum, magnesium and amine salts. In
the context of this invention, CMTAK includes not only its free
acid but also its salts, particularly those with low toxicity.
Table 3 shows the physical properties of some re-
presentative species of CMTAK which we prepared from TAK-N and
TAK-D by the procedure described hereinbefore. The determinations
of such physical properties were also made after each sample
was previously dried under reduced pressure and over phosphorus
pentoxide at 60~C for 10 hours.
,
- 10 -
~0~4453
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-
_ 1
~ - 1094453
1 Figs. 1 and 2 show the I.R~ spectra in potassium bromide
of CMTAK No. 2 and No. 11 in Table 3, respectively. Numerals
at the topside, the left side and the bottom side of each of the
figures indica-te wavelength (~), transmittance (%) and wave
number (cm~l), respectively.
The characteristic absorptions were found at 1440-1400
and 891 cm 1
The TAK-N, TAK-D and CMTAK obtained as above, ranging
from 2 to about 1,000, preferably from 15 to 800, more preferably
from 40 to 600 in DP, possess a significant inhibitory activity
against various kinds of tumors in warm-blooded animals including
domestic animals, poultry, dogs, cats, rabbits, rats, mice, etc.,
and particularly against solid tumors which are known to be less
sensitive to treatments with generally known cytotoxic antitumor
drugs.
For instance, the growth of sarcoma 180, Ehrlich
carcinoma, SN-36 leukemia, CCM adenocarcinoma, or NTF reticulum
cell sarcoma each transplated subcutaneously into mice was
markedly inhibited when TAK-N, T~K-D or CMTAK was administered
intraperitoneally, lntraveneously or subcutaneously, before,
after or at the time of transplantation, once or repeatedly at
dose levels of about 1 to 1,000 mg/kg per administration per day.
The toxicities of TAK-N, TAK-D and CMTA~ are extremely
low. For example, no toxic effect was observed both in mice and
rats at a dose of 3,000 mg/kg by intraperitoneal route or 10,000
mg/kg by oral route. Thus, these polysaccharides can be safely
administered to warm-blooded animals repeatedly.
The administration may be accomplished by general
procedures which are normally applied in the management of
cancers. Thus intra-tumor, subcutaneous, intramuscular, or
- 12 -
~0~44~
1 intravenous injections, if necessary, oral and rectal
administrations, or in external applications, coating,
instillation and other methods of administration are feasible.
According to the present invention, TAK-N, TAK-D or
CMTAK is administered in a sufficient amount to a warm-blooded
animal to inhibit growth of its tumor.
The dosage and procedure of the administration of TAK-N,
TAK-D or CMTAK to the warm-blooded animals affected by neoplasias
may vary according to the kind of agents ~TAK-N, TAK-D, CMTAK),
animals and patients, tumorous symptoms, administration forms
and other factors. Generally, the sufficient amount of dosage
per administration is about 0.02 to 2,000 mg/kg body weight,
preferably 0.2 to 2,000 mg/kg body weight, the preferred upper
limit being about 500 mg/kg body weight. In many cases, about
200 mg~kg is the most desirable upper limit.
The administration may be conducted, for example, one
to six times per day for consecutive or intermittent days.
The-form of in]ection is varied in each case but can
be realized in a per se conventional manner, that is to say,
TAK-N, TAK-D or CMTAK is dissolved or suspended in an aqueous
liquid medium directly or with a conventional solubilizer such as
alcohols (e.g. ethanol, propyleneglycol~, non-ionic surface
active agents, physiological saline and isotonic solutions.
In the case of CMTAK, the sample is easily dissolved in distilled
water or physiological saline.
For oral preparation, any member or pharmaceutical forms
can be used, for example, syrups, elixirs, suspensions or the
compound can be processed into wafers, pills, tablets supposi-
tories, gel and the like. In a preferred embodiment, the oral
dosage form consists of a tablet containing between about 10 and
- 13 -
10944S3
1 1,000 mg o the above active ingredient per tablet. Such
tablets can be coated in the usual fashion, preferably using a
readily soluble coating material, for example, sugar, etc.
The above amount can also be incorporated into gelatin capsules
which promptly dissolve upon introduction into the stomach.
In any event, the usual flavoring and coloring agents can be used
without effect on the active ingredient so incorporated.
Tablets of this type are prepared in the usual fashion
by compounding the active ingredient with a binding agent, e.g.,
starch, sugar and the like, granulating the mixture and, after
adding the necessary fillers, flavoring agents, lubricants, etc.,
the mixture is slugged and passed through a 30-mesh screen.
The thoroughly blended mixture is then compressed into tablets
of desired hardness with the usual punch, preferably to make
bisected tablets for easier administration.
TAK-N, TAK-D and CMTAK may be administered in combi-
nation with other antitumor agents. Combination with other anti-
tumor agents leading to the enhanced immunological activity of
the tumor-bearing animals is also desirable.
The invention will be further described by way of
examples. Some of these examples show the process of production
of TAK-D from TAK-N, the process of production of CMTAK, the
physical properties of TAK-N, TAK-D and CMTAK, and some of the
injectable forms of these agents. Other examples demonstrate the
antitumor activities of TAK-N, TAK-D and CMTAK against mouse
tumors by way of models.
As demonstrated above, the treatment of tumor-bearing
animals with the compounds produces drastic regression of
various kinds of tumors such as sarcoma, carcinoma, reticulum cell
sarcoma and leukemia. All of these types of tumors in rodents
- 14 -
~L0~ ~453
1 have been known to be reliable models for tumors in other warm-
blooded animals.
It should, o~ course, be understood that these examples
are by no means limitative of this invention.
~ ost of the following experiments on antitumor activity
of TAK-N, TAK-D, and CMTAK were tested by approximately the
same methods as those described for antitumor activity of
lentinan, a branched glucan from an edible mushroom, by Chihara,
G. et al. (See Nature 222, 687-688, 1969, and Cancer Research
30, 2776-2781, 1970).
Mouse tumors used in the experiments such as sarcoma 180
(S180), Ehrlich carcinoma (Ehrlich~, NTF reticulum cell sarcoma
(NTF), CCM adenocarcinoma ~CCM) and leukemia SN-36(SN-36) were
maintained in ICR-JCL mice, 5 to 7 weeks old, each in an ascites
form. A suitable number of these tumor cells was subcutaneously
transplanted into ICR-JCL mice, and the tumor nodes which had
grown up were excised for weighing on the 35th day after the
tumor transplantation.
A TAK-N or TAK-D, was suspended or dissolved in
distilled water or in physiological saline, and CMTAK was
dissolved in distilled water or in physiological saline in
adequate concentrations and at around neutral pH values. Each
preparation was administered intraperitoneally, subcutaneously or
intra~eneously to mice (0.2 ml/20g body weight) daily for 10 days
starting at 24 hours afer the tumor transplantation, daily for
5 days before tumor transplantation, or only once during the
7th day before and the 21st day after the tumor implantation.
EXAMPLE 1
Tumor Suppressive Effect of TAK-N (DP 540): Single Injection
Four millions of S 180 cells were subcutaneously trans~
~ 15 -
-- 109 ~S3
1 planted into the leEt in~uinal region oE ICR-JCL female mice,
4 to 5 weeks old. The tumor nodes were excised on the 35th
day after the transplantation, and weighed. The average tumor
weight of a treated group ¢T) consisting of 5 mice was compared
with that of control group (C) consisting of 10 mice, and the
tumor inhibition ratio (% Inhibition: (C-T)/C x 100) was
calculated. The number of tumor-free animals (complete
regression) on the 35th day was also counted.
TAK-N (DP 540) suspended in physiological saline
(10 mg/ml) was administered intraperitoneally (0.2 ml/20 g body
weight) once to each animal at the dose level of 100 mg/kg on the
7th, 3rd or 1st day before, or the 1st, 2nd, 3rd, 5th, 7th,
lOth, 14th, or 21st day after the tumor transplantation, or
on the day of tumor transplantation.
As shown in Table 4, TAK-N at the dose level of 100 mg/kg
inhibited the growth of S 180 significantly when the test
sample was administered only once during the 7th day before and
the 21st day after the tumor transplantation.
In addition, by a single administration of TAK-N
(DP 540) on the 7th day after the tumor transplantation at the
dose levels of 20 mg/kg, 60 mg/kg and 100 mg/kg, a significant
suppression of the tumor growth was observed at two higher
doses, 60 mg/kg and 100 mg/kg.
- 16 -
453
",
1 TABLE 4
Dose Day of Average Tumor No. of complete
Samp1 injection tumor inhibi- regression/No.
m(g/)g ratlot%)
----- 7.06 ---- 0/10
TAK-N 100 x 1 - 7* 2.27 67.8 3/5
(DP540 100 x 1-3 1.64 76.8 3/5
100 x 1 -1 0 100 5/5
~100 x 1 0** 0.56 92.1 3/5
~ 6.94 ---- 0/10
TAK-N 100 x 11*** 1.19 82.9 4/5
100 x 12 0.59 91.5 4/5
(DP540 100 x 1 3 0 100 5/5
100 x 1 5 1.25 82.0 3~5
100 x 1 7 0 100 5/5
----- 5.08 ---- 0/10
TAK - N (100 x 1 7 0 100 5/5
~100 x 1 10 0.01 99.8 4/5
(DP540 100 x 1 14 0.01 99.8 4/5
2~100 x 1 21 :1.71 66.3 1/5
_
----- 5.36 ---- 0/5
TAK - N r20 x 1 7 3.72 30.6 1/5
~ 60 x 1 7 0.12 97.8 3/5
(DP540)lloo x 1 7
,
* TAK-N was administered on the 7th day before the tumor
transplantation
** TAK-N was administered on the 1st day after the tumor
transplantation
*** TAK-N was administered on the day of tumox
transplantation
- 17 - -.,
~ 1094453
1 These results indicate that l'AK-N ~DP 540) is effective
in suppressing the tumor growth at all stages of tumor proyression
in warm-blooded animals even by a single administration.
EXAMPLE 2
Tumor Suppressive Effect of TAK-N (DP 540) and Autoclave-
curdled TAK-N (DP 540):
. . _ .
Six millions of S 180 cells were subcutaneously trans-
planted into the right inguinal region of ICR-JCL mice with body
weights of about 23 g. The tumor nodes were excised on the 35th
day after the transplantation, and weighed. The tumor
inhibition ratio in a treated group was calculated as in
Example 1.
TAK-N (DP 540) and autoclave-curdled TAK-N (DP 540)
were used. The latter was obtained by heating a 2 % suspension of
TAK-N (DP 540) at 120 for 25 minutes followed by drying and
powdering the formed gel. The samples were suspended in dis-
tilled water at desired concentrations, and each suspension was
administered intraperitoneally to a group of 5 mice daily for
10 days starting at 24 hours after the tumor transplantation.
As shown in Table 5, TAK-N ~DP 540) showed a pronounced
effect on the growth of tumors at the dose level from 1 to 50
mg/kg, the effect being particularly significant at the dose
level from 5 to 25 mg/kg.
The autoclaved TAK-N (DP 540)was also effective in tumor
growth suppression at the dose level of 10 mg/kg. There was
no observed toxicity.
- 18 -
lo~ ^C~3
1 TABLE 5
.
Dose Average Tumor No. of complete
Sample mg/~g tumor inhibition regression/No.
(ip) weight ratio (%) of treated mice
- 6.00 ----- 0/6
1 x 10 4.80 20.0 0/6
3 x 10 3.58 40.3 1/6
5 x 10 0.02 99.7 5/6
7 x 10 0.73 87.8 5/6
10 x 10 0.01 99.8 5/6
TAK-N
15 x 10 0.10 98.3 4/5
(DP 540)
20 x 10 0 100 5/5
25 x 10 0.42 93.0 3/6
50 x 10 1.19 80.2 1/6
TAK-N ----- 3.67 ----- 0/6
(DP 540) 10 x 10 0.29 92.1 5/6
* Autoclave-treated
These results indicate that TAK-N is useful in tumor
retardation at relatively low dose levels and that the sample
can be sterilized by autoclaving without loss of its activity.
EXAMPLE 3
Tumor Inhibitory Activity of Various Kinds _f TAK-N and TAK-D
.
From 24 hours after the transplantation of S 180 as
described in Example 2, various kinds of TAK-N and TAK-D suspended
in distilled water were administered in-traperitoneally at the dose
level of 10 mg/kg to a group of 5 mice daily for 10 days. The
tumor nodes were excised on the 35th day after the tumor
transplantation, and weighed. The inhibition ratios were cal-
culated as in Example 1.
- ~0~44S3
1 As shown in Table 6, all samples used exhibited an
inhibitory activity against the tumor growth. Especially,
those samples having DP over 50 showed a strong antitumor
activity with many complete regression.
TABLE 6
Dose Average Tumor No. of complete
Sample mg/kg tumor inhibition regression/No.
(ip) weight(g) ratio(~) of treated mice
~ 5.46 ---- 0/6
(DP 565) lOxlO 0.07 98.7 5/6
(DP 540) " 0.20 96.3 3/5
TAK-N (DP 380) 0.46 91.6 4/6
(DP 125) " 0 100 6/6
5.46 ---- 0/6
S-II
(DP 82) lOxlO 0 100 6/6
S-III
(DP 68) " 0.03 99.5 5/6
TAK-D F-III
(DP 50) " 0 100 6/6
F-V
(DP 39) " 2.03 62.8 1/6
F-VI
(DP 24) " 3.60 34.1 0/6
F-VII
(DP 16) ~ 1.54 71.8 1/6
.-
These results indicate that both TAK-N and TAK-D
are effective in tumor growth regression irrespective of their
DP values.
EXAMPLE 4
Tumor Suppressive Effects of CMTAK and Autoclave-treated CMTAK
The antitumor activity of three kinds of CMTAK prepared
from TAK-N (CMTAK Nos. 1, 4, 5) and of autoclaved CMTAK
- 20 -
~', .
10~ 4~;3
1 was assayed as described in Example 2. Samples were dissolved
in distilled water for injection, and the pH was adjusted to
around 7Ø In some experiments one of these samples (CMTAK No. 1)
was sterilized by autoclaving (120C, 25 minutes) before further
administration.
As shown in Table 7, all CMTAK samples at the dose
level of 10 mg/kg inhibited significantly the tumor growth.
These growth inhibition by CMTAK was observed at dose levels
from 1 to 40 mg/kg.
TAB~E 7
Dose Average Tumor No. of complete
Sample mg/kg. tumor inhibition regression/No.
~ip) weight(g) ratio(%) of treated mice
_--- 2.87
CMTAK No. 1lOx9 0 100 7/7
No. 4 lOx9 0.28 90.2 4/7
No. 5 lOx9 0.01 99.7 6/7
---- 3.65 ---- 0/6
~lxlO 0.77 78.9 1/6
Autoclaved
CMTAK No. 13xlO 0.03 99.2 5/6
5xlO 0.23 93.7 4/6
____ 2.17 ~~~~ 0!6
5xlO 0 100 6/6
I lOxlO 0.07 96.8 5/6
CMTAK No. 1~ 0.08 96.3 5/6
20xlO 0.21 90.3 3/6
l40xlO 0.18 91.7 1/6
These results indicate that CMTAK can be sterilized
without loss of its antitumor activity and that they are useful
in an injectable form for cancer treatment.
- 21 -
, ~
10~4453
1 EX~MPLE 5
Tumor Suppressive Effects of TAK-N, TAK-D and C~lTAK by various
Routes of Administration
TAK-N (DP 540), TAK-D (F-III: DP 50), TAK-D tF-VII:
DP 16), and CMTAK No. 1 were administered at the dose level of
5 mg/kg intravenously, subcutaneously or intraperitoneally for
10 consecutive days to ICR-JCL mice which were previously
transplanted with 4 x 106 cells at 24 hours before the first
injection. The inhibition ratio of tumor growth were calculated
as in Example 1.
As shown in Table 8, in each case, the significant
inhibition of tumor growth was observed. By intravenous adminis-
tration, TAK-N, CMTAK, and even F-VII (DP 16) inhibited the
tumor growth almost completely, the inhibition ratio being over
85 `% with the complete tumor regression in 4 or 5 out of 5 mice
treated.
TABLE 8
Dose Route of Average Tumor No. of com-
Sample(mg/kg) administra- tumor inhibi- plete re-
tion weight(g~ tion gression/No.
ratio(%) of treated
mice
----- 7.30 ----- 0/10
CMTAKJ 5 x 13 iv 0.8487.5 4/5
No. 1~ 5 x 10 sc 3.7748.4 0/5
~5 x 10 ip 0.9188.5 2/5
-
----- 7.30 ----- 0/10
TAK-N~5 x 10 iv 0 100 5/5
(~P 540)~ 5 x 10 sc 2.72 63 3/5
~5 x 10 ip 0.17 98 4/5
TAK-D~5 x 10 iv 3.02 59 2/5
F-III~ 5 x 10 sc 0.91 88 3/5
(DP 50)~5 x 10 ip 2~3a 68 3~5
30 TAK-D(5 x 10 iv 1.06 85 4/5
(DP 16)~ 5 x 10 sc 2.67 63 3/5
5 x 10 ip 2.34 68 2/5
,
. - 22 -
`` 10~44~i3
.
1 These results indicate that TAK-N, CMTAK and TAK-D
are e~fective in tumor suppression by any route of administration.
EXAMPLE 6
Tumor Su ressive Effects of TAK-N, TAK-D and CM~AK Administered
P P ~
Before Tumor Transplantation : Pre-treatment
TAK-N, TAK-D having different average degrees of po~y-
merization and CMTAK No. 1 were administered intraperitoneally
to ICR-JCL mice with body weights of about 23 g at dose level
from 3 mg/kg to 80 mg/kg daily for 5 consecutive days. TAK-N
and TAK-D were suspencled, and CMTAK was dissolved, in distilled
water. In the case of CMTAK the final pH was adjusted to
around 7Ø Then, on the 1st or 3rd day after the last
administration, 6 x 106 cells of S180 were transplanted sub-
cutaneously into the right inguinal region of each animal. On
the 35th day after the transplantation, the tumor nodes were
excised and weighed. Tumor inhibition ratios were calculated
as in Example 1. -
As shown in Table 9, a marked tumor growth inhibition was
observed by each sample. Especially TAK-N (DP 540), CMTAK and
TAK-D having DP over 50 exhibited a~strong inhibitory activity.
ClMTAK was effective for tumor retardation at dose level as low
as 3 mg/kg.
- 23 -
. .
453
1 TABLE 9
. _ . ... _ .
Dose Day of Average Tumor No. of complete
Sample mg/kg injection tumor inhibition regression/No.
(ip) _ _ weight(g) ratio(~) of treated mice
5.06 ----- 0/5
TAK-N
(DP 540) 20x5 -5 to -1 0.37 92.7 4/6
TAK-N
(DP 125) 20x5 -5 to -1 0.98 80.6 2/6
---- 10.10 ----- 0/6
S-II
(DP 82)20x5 -5 to -1 0.20 98.0 4/5
S-III
(DP 68)20x5 -5 to -1 0.35 96.5 5/6
F-III
(DP 50)20x5 -5 to -1 1.30 87.1 3/5
~--V
(DP 39)20x5 -5 to -1 5.52 45.3 0/6
F-VI
(DP 24)20xS -5 to -1 4.18 - 58.6 0/6
F-VII
(DP 16)20x5 -5 to -1 5.84 42.2 0/6
---- 6.43 ----- 0/6
~3x5 -5 to -1 1.44 77.6 3/5
~ 5x5 -5 to -1 3.55 44.8 1/6
CMTAK No.l lOx5 -5 to -1 0.19 97.1 3/6
20 20x5 -5 to -1 0.05 99.2 4/6
. .
5.03 ----- 0/5
CMTAK ~o 1~4X5 -5 to -1 0.11 97.8 4j6
~80x5 -5 to -1 0.15 97.0 4/6 -
... . . ~
7.45 ----- 0/5
TAK-N
(DP 540) 20x5 -7 to -3 0.18 97.6 3/5
TAK-D
S-III 20x5 -7 to -3 0.75 89.9 4/5
(DP 683
These results indicate that the polysaccharides mentioned
above are useful as prophylactics in cancer treatment.
- 24 -
10~ ~453
EXAMPLE 7
Tumor suppressive Activity o~ TAK-N and CMTAK against Various
Kinds of Tumors
Ehrlich carcinoma, CCM adenocarcinoma, NTF reticulwm cell
sarcoma and SN-36 leukemia were used to examine the usefulness
of TAK-N and CMTAK for the retardation of various kinds of
tumors.
ICR-JCL mice with an average body weight of 23 g were
transplanted subcutaneously with 3.1 x 106 Ehrlich cells, 1.1 x
107 CCM cells, 4.5 x 106 NTF cells or 7.2 x 104 SN-36 cells
and were administered intraperitoneally with TAK-N (DP 540)
suspended in distilled water or CMTAK No. 1 dissolved in
distilled water (pH 7.0) and autoclaved, at dose levels from
10 to 50 mg/kg daily for 10 days starting at 24 hours after the
transplantation. The tumors were excised and weighed 35 days
after the transplantation and the inhibition ratios of tumors
in the treated groups were calculated as in Example 1.
As shown in Table 10, in each case, a significant
~0 inhibition of tumor growth was observed.
- 25 -
~0~44~;3
1 TABLE 10
. _ _ _ _ _
Tumor Sample Dose Average Tumor No. of complete
mg/kg tumor inhibition regression/No.
_ _ _ (ip) weight(g)ratio(%) of treated mice
--- 3.54 --- 0/5
TAK-N ~lOxlO 2.37 33.1 0/5
Ehrlich(DP 540)~20xlO 0.50 85.9 , 2/6
carcinoma 40xlO 0.93 73 7 0~5
_____________________________ _____________ ____________
--- 1.93 --- 1/6
CMTAK J20xlo 0.80 58.5 3/6
No. 1 ~40xlO 0.60 68.9 2/6
TAK-N --- 3.42 --- 0/6
CCM adeno- (DP 540)20xlO 0.79 76.9 2/6
__________________________________________ ____________
carcinoma CMT~K 3.42 -__ 1/6
No. 1 20xlO 1.03 69.9 1/6
--- 0.91 --- 2/6
Leukemia TAK-N flOxlo 0.18 80.2 4/5
SN-36(DP 540)J25xlO 0.19 79.1 3/5
~50xlO 0.23 74.7 2/6
.. _ _ . ~ . ...
NTFTAK-N --- 7.64 --- 0/6
reticulum (DP 540)~10xlO 0.38 95.0 2/6
sarcoma~OxlO 2.21 71.1 0/6
__ .. . ...... . _ ~ _ . .
The results indicate that TAK-N and CMTAK have an
antitumor activity against several types of tumors, i.e.
sarcoma, carcinoma, adenocarcinoma, leukemia and so forth.
EXAMPLE 8
The four preparations of CMTAK (CMTAK No. 2, 6, 9 and 11)
were each dissolved in physiological saline and, according to
the procedure of Example 4, their effects of S180 tumor were
examined. All samples, at the daily dose level of 5 mg/kg per
- 26 -
~, -
10~4~53
1 administration for 10 days starting at 2~ hours after the tumor
transplantation, significantly suppressed the tumor growth.
EXAMPLE_9
TAK-N (replaceable by TAK-D or CMT~K)150 mg
Lactose 48 mg
Magnesium stearate 2 mg
Total200 mg
The above amount makes up one capsule.
TAK-N (TAK-D or CMTAK) and lactose are mixed in the
above-indicated proportions, tableted and pulverized. Then,
magnesium stearate is added. The mixture is dispensed into
capsules.
EXAMPLE 10
.. .. . _ _
TAK-N (replaceable by TAK-D or CMTAK 400 mg
Lactose 95 mg
HPC-L (hydroxypropyl cellulose) 5 mg
Total 500 mg
The above amount makes up a single dose.
The above three ingredients are mixed together in
the indicated proportions and with the addition of a small
amount of water, the mixture is kneaded in a kneader, granulated,
dried, regranulated, size-selected and packaged in doses
indicated above.
EXAMPLE 11
. .
(a) One gram of TAK-D (DP 16 ) is dissolved in 1,000 ml of
distilled water for injection (or physiological saline). The
solution is ~iltered and the filtrate is distributed into
ampoules in 500 ml portions. After sealing, the
ampoules are hea~-sterilized in a routine manner.
- 27 -
~0~9 ~453
(b) Two gram of CMTAK (the product of Example 14 is dissolvedin lO0 ml of distilled water for injection (or physiological
saline) and the solution is filtered. The filtrate is dis-
tributed into ampoules in 20 ml portions and, after sealing
by fusion, the ampoules are heat-sterilized in a routine manner.
EXAMPLE 12
TAK-N (replaceable by TAK-D or CMTAK) 160 mg
Sorbitol 200 mg
Sodium carboxymethylcellulose 10 mg
1 0
Polysorbate 80 3.2 mg
Methyl p-hydroxybenzoate 4 mg
Propyl p-hydroxybenzoate 0.4 mg
The above ingredients are mixed in distilled water for
injection to make a total volume of 4 ml. ~When CMTAK is
employed, the solution is neutralized, if necessary, with
N/10 - sodium hydroxide)
EXAMPLE 13
In 80 ml of isopropyl alcohol was suspended 3 g of
TAK-N (DP 5~0) and the suspension was stirred at room temperature
for 30 minutes. Then, 8 ml of a 30~ solution of sodium hydroxide
was slowly added with stirring over a period of about 60 minutes.
The mixture was further stirred vigorously at room temperature
for about 90 minutes for the purpose of preventing the formation
of a gel. Then, 3.6 g of monochloroacetic acid was added, and
the mixture was stirred at 60 - 70C for 5 hours to allow the
carboxymethylation to proceed. The product was recovered by
filtration and thoroughly washed with a mixture of methanol and
acetic acid (7 : 3, v/v). The precipitate was collected by
filtration, washed well with 80 ~ aqueous methanol, methanol and
- 28 -
1094~3
1 acetone in the order mentioned and dried under reduced pressure.
By the above procedure was obtained 2.9 g of CMTAK. Car~oxymethyl
content (the number of carboxymethyl group per glucose residue;
the same definition also applies hereinafter): 0.54.
EX~PLE 14
In 40 ml of isopropyl alcohol was suspended 1.5 g of
TAK-N (DP 540) and the suspension was stirred at ro3m temperature
for 30 minutes. Then, under stirring, 2 ml of a 30 % solution
of sodium hydroxide was added in four installments, i.e. 0.5 ml
each at intervals of 15 minutes. The mixture was further
stirred at room temperature for 90 minutes. Then, 0.9 y of
monochloroacetic acid was added in three installments, i.e. 0.3 g
each at intervals of 10 minutes. The carboxymethylation was
thus conducted at 50C with stirring for 150 minutes. The product
was collected by centrifugation, dissolved in 50 ml of water
and neutralized with acetic acid. To this neutral solution was
added 120 ml of methanol and the resulting precipitate was
collected by centrifugation. The precipitate was washed with
a mixture of 300 ml of 80 % aqueous methanol and 100 ml of
:20 ''
ethanol and, then, with a mixture of 300 ml of 80 % aqueous
methanol and 200 ml of ether. The product was finally
lyophili2ed to recover 1.7 g of CMTAK. Carboxymethyl content :
0.75.
EXAMPLE 15
. .
In 40 ml of isopropyl alcohol was suspended 1.5 g of
TAK-N (DP 540) and the suspension was stirred at room temperature
for 30 minutes. Then, 4 ml of a 30% solution of sodium
hydroxide was added in 4 installments, 1 ml each at intervals
of 15 minutes, followed by stirring at room temperature for
90 minutes. Thereafter, 1.8 g of monochloroacetic acid was added
- 29 -
109~453
1 in 3 installments, i.e. 0.6 g each at intervals of 10 minutes.
The mixture was stirred at 50C for 150 minutes, whereby the
carboxymethylation was allowed to proceed. The praduct was
collected by centrifugation, dissolved in 40 ml of water and
neutralized with acetic acid. To this neutral solution was
added 90 ml of methanol and the resulting precipitate was
collected by centrifugation. The precipitate was washed well
with a mixture of 200 ml of 80 % aqueous methanol and 100 ml
of ethanol and, then, with a mixture of 200 ml of 80 % aqueous
ethanol and 200 ml of ether. It was then lyophilized to
recover 2.0 g of CMTAK. Carboxymethyl content: 1.07.
EXAMPLE 16
.
In 33 ml of water was suspended 3.2 g of TAK-N (DP 255)
and, under stirring at room teperature, 1 g of sodium hydroxide
was added, followed by addition of 2.4 g of sodium monochloro-
acetate. The carboxymethylation reaction was thus conducted
at room temperature with constant stirring for 2 hours. There-
after, 1 g of sodium hydroxide and 2.4 g of sodium monochloro-
acetate were added again and the reaction was further conductedat room temperature with stirring for 3 hours. Then, 1 g of
sodium hydroxide and 2.4 g of sodium monochloroacetate were
further added. The reaction was continued at room temperature
with stirring for an additional 2 hours. To the reaction mixture
was added 1 liter of ethanol and the resulting precipitate
was washed well with ethanol on a glass filter until the filtrate
ceased to give a red color with phenolphthalein. It was then
dried at 50C and under reduced pressure. The resulting powder
(3.7 g~ was dissolved in 90 ml of water and neutralized with
acetic acid, followed by addition of 210 ml of ethanol. The
resulting precipitate was collected by centrifugation, washed
30 -
109~4S3
with 80 % aqueous ethanol and lyophilized. By the above
procedure was obtained 2.6 g of CMTAK. Carboxymethyl content:
0.30.
EX~MPLE 1 7
In 66 ml of water was suspended 6.4 g of TAK-N (DP Z55)
and, under ice-cooling and stirring, 4 g of sodium hydroxide
and, then, 9.6 g of sodium monochloroacetate were added. The
carboxymethylatlon was conducted under ice-cooling and stirring
for 2 hours. Then, 4 g of sodium hydroxide and 9.6 g of sodium
monochloroacetate were added again and the mixture was stirred
under ice-cooling for 3 hours. Thereafter, 4 g of sodium
hydroxide and 9.6 g of sodium monochloroacetate were further
added and the reaction was continued under ice-cooling and
stirring for 3 hours. To the reaction mixture was added 1 liter
of ethanol and the resulting precipitate was washed well with
ethanol on a glass filter until the filtrate ceased to give a
red color with phenolphthalein, followed by drying at 50C
and under reduced pressure. The resulting powder (~.6 g) was
dissolved in 172 ml of water and neutralized with acetic acid.
Then, following the addition of 480 ml of ethanol, the pre-
cipitate was collected by centrifugation, washed with 80 %
aqueous ethanol and lyophilized. By the above procedure was
obtained 5.6 g of CMTAK. Carboxymethyl content: 0.36.
EXAMPLE_18
In 40 ml of isop~opyl alcohol was suspended 1.5 g of
TAK-D (F-I, DP 299) and the carboxymethylation was conducted as
in Example 14. The product was washed as in Example 14 to
obtain 1.9 g of CMTAK. Carboxymethyl content: 0.59.
The same TAK-D as above was suspended in 40 ml of isopropyl
10~ ;3
1 alcohol and, by the same procedure as that described in
Example 15, the carboxymethylation was carried out. The reaction
product was washed as in Example 15 to recover 2.2 g of CMTAK.
Carboxymethyl content: 1.15.
EXAMPLE 19
. .
In 40 ml of isopropyl alcohol was suspended 1.5 g of
each of TAK-D (F-II, DP 113) and TAK-D (S-III, DP 68),
respectively. And, as in Example 14, the carboxymethylation
was carried out. The respective reaction products were collected
by centrifugation, dissolved in 40 ml of water and neutralized with
acetic acid. To each of these two neutral solutions was
added 90 ml of methanol and the resulting precipitate was
collected by centrifugation and washed well first with 200 ml
of 80 % aqueous methanol and then with 200 ml of aqueous
ethanol, followed by lyophilizing. By the above procedure were
obtained two species of CMTAK.
Carboxymethyl
Yield content
CMTAK (obtained from F-II 1.4 g 0.51
" ~ " from S-III) 1.4 g 0.36
EXAMPLE 20
In 40 ml of isopropyl alcohol was suspended 1.5 g of
each of TAK-D (F-II, DP 113~ and TAK-D (S-III, DP 68), res-
pectively and, as in Example 15, the carboxymethylation was
carried out. The reaction products were washed respectively as
in Example 19 to recover two species of CMTAK.
Carboxymethyl
Yield content
30CMTAK (obtained from F-II) 2.3 g 1.22
" ( " from S-III) 1.4 g 0.45
- 32 -
.
