Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
110~876
This invention relates to the use of a nitrogen-
containing polysaccharide for promoting the drug sensitivity
of bacteria which have become resistant to antibiotics.
Advancement of chemotherapeutical techniques and,
particularly, the pervasion of a variety of antibiotics have
realized a drastic reduction of bacterial infectious diseases
such as tuberculosis, dysentery, etc., and their contribution
to public health is beyond description. However, these modern
achievements have invited, on the other hand, a rapid increase
of so-called drug-resistant bacteria which defy the existing
chemotherapeutants. To make the matter worse, many "multiple-
drug-resistant bacteria", that is, bacteria resistant to two or
more kinds of drugs, have developed from dangerous serms such
as Mycobacteria, Shigella, Staphylococci, etc., and these
troublesome newcomers are posing a serious problem in the treat-
ment of infectious diseases.
For this reason, antibiotic preparations usually have
a short life cycle and hence the drug manufacturers are compelled
to market new kinds of antibiotics one after another in rapid
succession at an enormous cost. This can result in a huge loss
which cannot be ignored from the viewpoint of social economy.
With the aforesaid situation in mind, we have pursued
extensive studies of drug-resistant bacteria and, as a result,
have found that the use of a specific nitrogen-containing poly-
saccharide in combination with an antibiotic can bring about
a striking enhancement of drug sensitivity of germs which have
become resistant to the antibiotic. It has also been found that
the nitrogen-containing polysaccharide is able not only to
enhance the potency of the antibiotic against the resistant
germs but also to expand its effect to allow long-term use of
the antibiotic.
-- 2 --
11011876
According to the invention there is provided
a pharmaceutical composition comprising (a) a nitrog~n-
containing polysaccharide obtainable by extracting mycelia
of basidiomyceto~s fungus belonging to the genus Coriolus
of Polyporacease with an aqueous solvent and (b) an anti-
biotic, for use in treatment of humans and mammals suffer-
ing from a disease caused by a strain of bacterial species,
which strain has developed its resistance to said anti-
biotic, characterized in that said polysaccharide has a
molecular weight of from 5,000 to 300,000 as determined by
ultracentrifugation, contains 42.0 to 46.0% of carbon, 5.3
to 7.0% of hydrogen, 0.5 to 8.0~ of nitrogen and the bal-
ance of oxygen, has a specifiec rotatory power of 0 to
+50.0, is easily soluble in water but insoluble in ace-
tone, prridine, chloroform and hexane and shows character-
istic absorptions at 840 and 890 cm 1 in the infrared
absorption spectrum and has a ratio of saccharide portion
to protein portion of 62/38 to 97/3 as determined by nuc-
lear magnetic resonance spectroscopy, and said antibiotic
being selected from the group consisting of streptomycin,
chloramphenicol, penicillin G. kanamycin, aminobenzyl-
penicillin, tetracycline, erythromycin, cephaloridine and
collistin.
The invention, and particularly preferred embodiments
thereof, are described in detail below with particular reference
to the accompanying drawings, in which:-
Figure 1 is an infrared absorption spectrum of the
nitrogen-containing polysaccharide (NP~ according to this
invention;
Figure 2 is a proton nuclear magnetic resonance
absorption spectrum (N.M.R.) of the substance; and
Figures 3 to 6 are the graphs showing the effects
obtainable according to the present invention.
~.c~l - 3 -
I
1~0~876
The nitrogen-containing polysaccharide which is used
as an active component in this invention is obtainable by
extracting the mycelia of the basidiomycetes belonging to
the genus Coriolus of Polyporaceae with an aqueous solvent.
The term "mycelia of basidiomycetes belonging to the genus
Coriolus of Polyporaceae" as herein used is based on the
classifieation in "Coloured Illustration of Fungi of Japan" by
Rokuya Imazeki and Tsugio Hongo ~Hoikusha Pub. Co.).
The method of preparing the nitrogen-containing
polysaceharide used as the aetive eomponent in this invention
is briefly deseribed below.
The mycelia of basidiomycetes belonging to the genus -
Coriol_ , which are used as the starting material for the
extraction process can be produced either naturally or by an
artifieial culture. In the case of artifieially eulturing
the fungi by using a liquid medium, it is possible to use as
the starting material all the eulture produet ineluding not
only the myeelia thus produeed but also the eluate in the
liquid medium after the eulture. The thus obtained starting
material is extraeted with an aqueous solvent and the extraet
is subjected to suitable treatments such as filtration, neutrali-
zation, concentration of the filtrate, dialysis, salting-out,
ete. to remove the low-moleeular material (i.e. material
with a moleeular weight of lower than 5,000) from the extraet,
..
llOU87~i
¦¦ and is dried to obtain a powdery substance. The thus obtained
powdery substance is composed of a nitrogen-containing
Il polysaccharide which can be used as the active component in this
¦l invention.
1ll The nitrogen-containing polysaccharide obtained in
! the manner described above has the following properties.
Physiochemical properties:
(1) General
~ The substance is powdery in form and brown in color,
I shows no definite melting point and is carbonized when strongly
I heated. It is insoluble in the organic solvents such as pyridine,
¦¦ chloroform, benzene, hexane, etc., but soluble in water.
¦ (2) Infrared absorption spectrum
The infrared absorption spectrum of this substance
indicates absorption at and in the vicinity of 3,600 - 3,200 cm 1,
2,920 - 2,900 cm 1, 1,660 - 1,610 cm 1, 1,460 cm 1, 1410 cm 1,
¦l 1,360 cm 1, 1,230 cm 1, 1,150 cm 1, 1,080 cm~l, 1,060 - 990 cm 1,
¦l 925 cm 1, 890 cm 1, 840 cm 1, 755 cm 1 and 705 cm 1. Thus,
,'l there are noted absorptions by the ~-bonds of glucan in the
iI saccharide portion at 890 cm 1 and another absorption by the
Il ~-bonds of glucan at 840 cm 1.
¦¦ (3) Elementary analysis
The composition of the substance, as measured by an
~1 elementary analysis, comprises 42 to 46~ of carbon, 5.3 to
ll 7.0% of hydrogen and 0.5 to 8.0% of nitrogen. The balance is
oxygen.
Il
!l
- 5 -
l l
,1 1 .
ll i
0~76
I
. I
~4) Optical rotation
The optical rotation of the substance, as determined
in terms of the specific rotatory power [~]D is 0 to 50.
!i (5) Color reaction
5 1l The substance is positive in the phenol-sulfuric acid
! reaction, anthrone-sulfuric acid reaction and ninhydrin reaction.
! The positive disposition in these color reactions indicates
¦I that the active substance used in this invention is composed
I of saccharide and protein. In order to determine the saccharide
! composition of the nitrogen-containing polysaccharide, a sample
was hydrolyzed with methanolic hydrochloric acid and, after
trimethylsilylation by a known method, subjected to gas
chromatography. The result showed that said substance is
I mainly composed of glucose and also contains mannose, galactose,
, xylose and fucose. An amino acid analysis of the protein
portion of the nitrogen-containing polysaccharide revealed that
! the protein portion of the substance is composed of aspartic
j, acid, threonine, serine, glutamic acid, proline, glycine, alanine,
,I cystine, valine, methionine, isoleucine, leucine, tyrosine,
!I tryptophan, phenylalanine, lysin, histidine, and alginine.
Predominant among them are aspartic acid, threonine, glutamic
acid, glycine, alanine, valine and leucine, and they account
! for more than 70% of the entire amino acids.
Il~ (6) Proton nuclear magnetic resonance spectrum (~R)
11 An NMR absorption spectroscopy was carried out for
,, , I
.
1101~876
¦ determining the ratio between the saccharide and protein
portions in the nitrogen-containing polysaccharide as well as
¦I the saccharide bonds therein.
Il The NMR absorption spectrum of the sample was measured
¦1 at 100 MHz by using heavy water as the solvent and sodium
¦l 2,2-dimethyl-2-silanopentane-5-sulfonate (D.S.S.) as the internal
¦¦ standard. As shown in Fig. 2, absorptions are noted at 0.9+0.2 -
¦l 1.2+0.2 ppm, 2.0+0.2 ppm, 4.5+0.2 ppm, 4.7+0.2 ppm, 5.0+0.2 ppm
I and 5.4+0.2 ppm, respectively, and a broad absorption band
I is seen at 3 0 - 4.4 ppm. The protein portion was examined by
assuming that the region of 0.5 - 2.5 ppm is associated with
the proton intensity of the protein portion and the region of
2.5 - 6.0 ppm is associated with the proton intensity of
saccharide. The protein portion was estimated at less than
1 40%. Also assuming that the region of 4.4 - 4.9 ppm is
associated with the ~-bonds of the saccharide and the region
1~ of 4.9 - 5.4 ppm is associated with the a-bonds, their ratio
/a) was determined to be within the range of 85/15 to 40/60.
,l (7) Molecular weight
¦~ The molecular weight of the nitrogen-containing
polysaccharide in this invention, as measured by ultracentrifu-
j gation, was within the range of 5,000 to 300,000.
Acute toxic ~
IlA test was carried out on mice of the ICR/JCL strain,
4 to 5-weeks old, weighing 21 to 24 g and on rats of the Do-ryu
~ 7 _
il. .
1101~876
strain, 4 to 5-weeks old, weighing 100 to 150 g. The substance
' dissolved in a physiological salt solution was administered
via the following four routes: intravenous, subcutaneous,
I intraperitoneal and oral. The general symptoms, the death rate
1~ and the change of body weight of the test animals were observed
for a period of 7 days, and thereafter they were killed and
, autopsied. As a result, no deaths occurred in both the
rats and mice even at the highest dose as shown in Table 1
¦ below. Determination of LD50 was there~ore practically impossible
I because none of the animals died in the experiment.
I Table 1
Animal Route of administration LDso (mg~kg) ~- -
Female Male
I _ ._ _ _ _ _ _ _ __l Intravenous >1,300 >1,300
~, Subcutaneous ~ >5,000 >5,000
!~ Mouse
Intraperitoneal >5,000 >5,000
Oral >20,000 >20,000
i .. .. . ..
il Intravenous >600 >600
l, Rat Subcutaneous >5,000 >5,000
li Intraperitoneal >5,000 >5,000
I oral >20,000 >20,000
l l l ~
! ~
.
, -~ 8 -
~. :-
'` ~.
I1 110~876
I
We will now describe the.effect of the nitrogen-
containing polysaccharide (hereinafter referred to simply
as the "present substance") for promoting the drug-sensitivity
, of drug-resistant bacteria which have become resistant to
I, antiobiotics.
Examples of the antibiotics which exhibit their effect
¦l against the drug-resistant bacteria when combined with the pre-
' sent substance are those derived from molds, bacteria, actinomyces
il and the like, and typical examples of such antibiotics
lQ ¦l are as listed below:
Penicillin G ~hereinafter abbreviated as PG)
li Streptomycin (hereinafter abbreviated as SM~
! Kanamycin (hereinafter abbreviated as KM)
1ll Chlorampheniocol (hereinafter abbreviated as CP)
15 1~ Tetracycline thereinafter abbreviated as TC)
Erythromycin ~hereinafter abbreviated as EM)
,, Aminobenzylpenicillin (hereinafter abbreviated as ABPC)
Cephaloridine (hereinafter abbreviated as CER)
Il Colistin (hereinafter abbreviated as CL)
'¦ The present substance promotes the drug-sensitivity
lof the following drug-resistant bacteria to the antibiotics:
¦ Escherichia coli
Il Streptococcus faecalis
I Staphylococcus aureus
I'
lll
-- g _
,'' .
,
~1
1101~876
ll
Enterobacter aerogenes
Salmonella enteritidis
Shigella Sonnei
I Xlebsiella pneumoniae
I Proteus mirabilis
Pseudomonas aeruginosa
¦I The effect of the present substance in promoting the drug
sensitivity of the drug-resistant bacteria was confirmed in the
Il following way.
¦I The drug-resistant bacteria were obtained in the manner
¦! described below by the concentration gradient plate
¦I culture (see "Chemotherapeutics and Resistant Bacteria",
1970, by Chikara Watanabe, Asakura Shoten).
¦l Agar plates with drug concentration gradients
ll ranging from 10 to 100 ~g/mQ were prepared, and the bacteria of
each strain to be tested were streaked or smeared thereon. These
¦li plates were kept at 37C for several days and the colony formed
in the high concentration area was isolated and again similarly
ll inoculated onto the plates. This operation was repeated several
~I times to obtain the bacterial strains resistant to the respective
drugs.
The drug sensitivity of the drug-resistant strains
and that of the original strains (sensitive strains) were
¦! compared by way of the minimum growth inihibiting concentration
25 li (hereinafter abbreviated as MIC) according to the Standard
¦I Method of the Japanese Chemotherapeutical Association (see
ll
.,..'
lll
- 10
.
!
.
- , ~
876
l ~
¦ "Chemotherapy" Vol. 22, No. 6, 1126 (1974), by MIC Determination
Method Reform Committee). More specifically, there were
prepared double diluted systems of each drug and they were mixed
~, with the heart infusion agar medium (Nippon Eiyo Kagaku Co., Ltd.)
5 1l to form the agar plates. Then a loopful of each strain, which
had been cultured at 37C in Trypto-soy bouillon (Nippon
Eiyo Kagaku Co., Ltd.~ for 18 hr. was smeared on each plate,
and after culturing for 18 hr at 37C, the growth of the strain
Il on each plate was examined.
10 ll In order to see the change of MIC for each resistant
strain by the combined use of the present substance and the
antibiotics (those which have lost their effect on the bacterial
strains), the same procedure was carried out as above
except for admixing, in addition, 10 to 1,000 ~g/mQ
I of the present substance into the systems and the MIC value
was determined according to the afore-mentioned agar plate
method.
The MIC has apparently been reduced to half the level
I before addition or less by the addition of the present substance.
1I The amount of the present substance added is advantageously
more than 10 ~g/mQ, preferably more than 100 ~g/mQ. The pH
value of the medium was maintained at 7.2+0.1 so that the MIC
would not be affected by the pH of the mRdium. P.lso, it
! was previously ascertained by the agar dilution culture
1 method that the present suhstance per se has no anti-
bacterial activity against the bacterlal strains tested.
Il
I I - 1 1 --
.1
,. - i
8~/~
¦ Therapeutical test for infectious~diseases:
¦ The therapeutical test for infectious disease was
carried out in the following way.
lll A mouse was intraperitoneally inoculated with 1 x 108
¦¦ cells of the drug~resistant bacteria, and 1 to 3 hours later, 10
to 1,000 mg/kg (body weight of mouse) of the drug and 1 to 104mg/kg
¦¦ of the present substance were administered either intraperitoneally
or orally. Daily observation was continued for 7 days after
the administration to examine the rate of survival for evaluating
the effectiveness of the present substance. In this case, it
was found that the effective dosage of the present substance was
preferably over 10 mg/kg for intraperitoneal administration
and over 100 mg/kg for oral administration. It was higher than
40%.
Thus, it was ascertained that the present substance
¦ is capable of enhancing the therapeutical effect not only in
¦¦ invitro applications but also in chemotherapy of the infectious
diseases tested.
! The present substance also shows an extremely low acute
¦ toxicity and it can be administered in various ways, such as
intraperitoneal injection, dermal administration, oral adminis-
tration and intrarectal administration. Thus, the present
substance may be mixed with an antibiotic in the course of
drug preparation or may be administered singly.
When the substance is prepared into tablets, granules,
~;
. ~ - ' . -
110(,!876
powder, capsules or the like for oral administration, the
composition of any such preparation may contain the additives
of the type generally used in drug preparation, such as a binding
¦ agent, inclusion agent, excipient, lubricant, disintegrator,
li wetting agent~ etc. When the substance is used as a liquid
for oral administration, it may be prepared into the form of an
¦ internal liquid medicine such as a shake mixture, suspension,
j emulsion, sirup, etc., or it may be prepared into the form of
~ a dry product which is re-dissolved just before use. Such
¦ liquid preparations may also contain the additives and~or
¦ preservative of the type usually used for drug preparations.
¦¦ The injections may contain the additives such as stabilizer,
¦¦ buffer, preservative, isotonizer, etc., and may be supplied
li in the form of unit dose ampules or multiple dose containers.
¦l Also, the above-said compositions may be provided in the form
of an aqueous solution, suspension, solution or emulsion
, in an oil or aqueous vehicle, while the active component may be
given in the form of a powder which is redissolved with a
Il suitable vehicle, for example sterilized pyrogen-free water,
l just before use. In case the substance is used in the form of
an ointment or inunction, it may contain an oil or fat base,
emulsive base, water-soluble base, preservative or the like.
The invention is now described by way of Examples
¦' thereof to further clarify the outstanding effects of the
invention.
Il
ll
ll
- 13 -
~,,,
1,1 ' .
.,
1100876
.~
Il
Il EXAMPLE 1:
I
I Preparation of nitrogen-containing polysaccharide:
il .
A liquid medium of the following composition was prepared:
Il Peptone 5 g
5 I Yeast extract 3 g
2 4 0.3 g
2 4 0.3 g
MySO4 7H2O 0.3 g
Glucose 50 g
Water 1 litre
pH: 6.0
An aliquot of 150 mQ of this medium was introduced
into each of 100 conical flasks of lQ capacity, and
after sealing each flask with a cotton plug, the liquid medium
was subjected to sterilization for 30 min at 120C. and then
inoculated inllthe usual way with the separately slant-culturedl
li mycelia of Coriolus versicolor ~Fr.~ Quel. CM-105 strain (Ferm.
Il ..
¦¦ Res. Inst. Deposit No. FERM-P-2416) followed by a 20-day stationary
¦¦ culture at 25 to 27C. The thus obtained culture slurry (broth)
¦ was dried by a double-drum type drum-dryer to obtain 451 g
of a dry product. One hundred and fifty grams of this dry
product was crushed and extracted with 0.lN sodium hydroxlde
solution at a temperature of 95 - 98C. and under normal
l pressure for 3 hr by using a stainless extractor. The thus
¦ obtained alkaline extract solution was neutralized and filtered
and then the filtrate was concentrated to 500 ml. This
concentrated solution was then encapsulated in a cellophane
¦!
: ~'
~ - 14 -
,1
1, ' .
B
llU(~876
film and subjected to dialysis in~ running water for 90 hr.
The obtained dialyzate was further concentrated under
l reduced pressure and spray-dried to obtain 19.5 g of powdery
Il product.
¦ In order to examine the properties of this powdery
I product, a sample thereof was subjected to an elementary
¦¦ analysis by using a CHN-CORDER MT-2 analyzer (Yanagimoto Seisakujo
Co., Ltd.), the result showing 42.1% of carbon, 6.9% of hydrogen
and 5.8% of nitrogen. Determination of specific rotatory
power was carried out on a 0. 25% aqueous solution of the sample
by using a YANACO-OR-50 Model (Yanagimoto Seisakujo Co., Ltd.).
The result showed the specific rotatory power of this product
is ~12.
In order to determine the saccharide composition of the
product, 10 mg of the sample was added to 3% methanolic
! hydrochloric acid to perform methanolysis for 16 hr at 100C.
¦I The reactant was filtered after neutralizing the hydrochloric
l acid with silver carbonate at room temperature, and the obtained
! filtrate was concentrated and evaporated to dryness. The
20 1I solid product was then dissolved in 0.5 ml of pyridine and
¦ further added with 0. 2 ml of hexamethyldisilazane and 0.3 ml
of trimethylchlorosilane, the mixture being allowed to stand
¦ at room temperature for 30 min to effect trimethylsilylation.
¦ The product was then dissolved in chloroform, and after washing
1 away excess reagent and dehydrating, the filtrate was evaporated
I!
- 15 ~
I,
!i i
0(~876
¦ to dryness. This product was theh dissolved in carbon tetrachlo- ¦
j ride and subjected to gas chromatography. The result showed
70.5% of glucose, 3.8% of galactose, 10.3% of mannose, 5.4~ of
xylose and 10.0% of fucose.
I For determining the protein composition of the
! powdery product, a sample thereof was subjected to an amino
¦ acid analysis according to a normal method. The result showed
¦ the following composition: 15% of aspartic acid, 8% of threonine,
6% of serine, 14% of glutamic acid, 4% of proline, 8% of glycin,
10% of alanine, 4% of isoleucine, 6% of leucine, 1% of tyrosine,
4% of phenylalanine, 2% of tryptophan, 2% of lysin, 3% of
¦¦ alginine, 4% of ammonia and 1~ N-glucosamine, and a trace of
i histidine.
¦ As for NMR absorption, heavy water was used as a
¦I solvent while employing DSS as an internal standard, and in order
to eliminate any possible influence of residual light water
¦l in the heavy water, the values which had undergone cor-
rection based on the presumed Lorenz's curve were used.
,l Under these conditions, the ratio of saccharide portion to
¦i protein portion was determined by assuming that absorption at
¦ 0.5 - 2.5 ppm is due to proton in the protein portion and
absorption at 2.5 - 6.0 ppm is due to proton in the saccharide
portion. The obtained ratio of saccharide to protein was
¦¦ 89/11. Also, the ratio of ~/~ was determined by assuming that
an absorption band at 4.4 - 4.9 ppm is associated with
- 16 -
!
'I , ,
ll l
110~876
¦ the ~-bonds of saccharide and an absorption band at 4.9 - 6.0
¦! ppm is associated with the ~-bonds. The ratio was 65/35.
¦ The molecular weight was determined by means of
¦l ultracentrigugation, and it was carried out by employing
¦ the sedimentation equilibrium and synthetic boundary pattern
utilizing an interference optical system under the following
~¦ conditions: concentration of sample of 0.3%; solvent of M/10
KCl; temperature of 25C.; liquid column of 1.7mm; and rotation
of 22,000 r.p.m. with a measuring time of 5 hr.
The obtained average molecular weight was 100,000.
Test of the effect of promoting the drug-sensitivity of
¦ the drug-resistant bacteria, which have become resistant to
antibiotics:
I (1) Preparation of drug-resistant bacteria
I Specimens of the drug-resistant bacteria were obtained
¦l aecording to the afore-mentioned eoneentration-gradient plate
eulture by using Staphyloeoceus aureus, Streptoeoeeus faeealis,
Escherichia eoli, Salmonella enteritidis, Klebsiella pneumoniae
Il and Pseudomonas aeruginosa. The MICs of the original
I baeteria and those of baeteria whieh have developed resistanee
to the drags were summarized in Table 2 below.
- 17 -
,
llOU876
ll Table 2
_ .. . . ~
Bacteria Antibiotics Minimum growth inhibiting
concentration._MIC (~g/mQ) _
Original Resistant
strain strain
l .~ . . .__ .. _
., Staphylococcus PC 0.025 12.5
i aureus CP 3.1 25
TC 0.8 25
; EM 0.4 12.5
I SM 1.6 100<
¦ Streptococcus TC0 4 25
faecalis
Escherichia
coli PC12.5 100<
SM3.1 100
~l6.3 200
CP1.6 50
TC3.1 25
CER3.1 25
EM100< -
Enterobacter ~l3.1 100
aerogenes
¦¦ Salmonella PC6.3 loo<
lenteritidis CP0.8 12.5
1 SM25 100<
: ; TC1.6 12.5 .
i EM100 < -
.' 11
. i Shigella KM3.1 50
; I sonnei
, Klebsiella PC6.3 100
¦pneumoniae SM1.6 100
i CP0.8 25
TC1.6 25
ll EM12.5 100
35 I Proteus mirabilis ABPC0.8 25
Pseudomonas CL12.5 200
aeroginosa
I .
¦¦ The MIC values of PC were calculated on the assumption that
ll 1,667 U (units) = lmg;
1 The MIC values of CL were calculated on the assumption that
l 30,000U (units) = 1 mg
.1 ~ '
- 18 -
ll
!!
110~876
Il (2) Effect of promoting the drug-sensitivity of the drug-
resistant bacteria
In order to determine the change of MIC value of the
¦ respective drug-resistant strains by the joint use of the present ¦
I substance and various antibiotics (those used for providing
the bacteria with resistance), the present substance was
added to the culture medium in amounts of 10 to 1,000 yg/mQ
and the MIC values were obtained from the agar plate method.
The r3sults are sbown collectively in Table 3 below.
.
I! .
ll
1 - 19 - '
I' , I
1. 1
oa~76
~ Table 3
_ .
Resistant Drug Concentration Minimum growth Inhibiting
,! strain (antibiotics) of present concentration
of bacteria subs~ance (~g/mQ)
t~g/mQ) ~ot-added ** Added*
_ _ _ __
¦ Staphylococcus
¦ aureus
j PC-resistant PC 1000 12.5 1.6
CP " CP 100 25 6.3
TC " TC 100 25 3.2
EM " EM 1000 12.5 3.2
Streptococcus
faecalis
TC-resistant TC 100 25 6.3
Escherichia
coli
SM-resistant SM 1000 100 25
KM " KM 1000 200 50
CP " CP 100 50 12.5
TC " TC 100 25 6.3
OE R " CER 10 25 12.5
Enterobacter
aerogenes
KM-resistant KM 1000 100 12.5
1' Salmonella
jj enteritidis
CP-resistant CP 100 12.5 3.2
TC " TC 100 12.5 6.3
,j Klebsiella
30 I pneumoniae
SM-resistant SM 1000 100 25
CP " CP 100 25 6.3
TC " TC 100 25 12.5
Shigella
¦ sonnei
KM-resistant KM 1000 50 . 12.5
. Proteus
mirabilis
l ABPC-resistant ABPC 10 25 12.5
1 Pseudomonas
aeruginosa
CL-resistant CL 1000 200 100
..~
Notes: * The present substance was added to the culture
medium
** The present substance was not added to the
, culture medium
- 20 -
l! I
. .. il
I
~1 11()~876
l I
EXAM~LE 2
_ ~
A therapeutical test for infectious diseases was carried
~¦out on five groups of ICR-JCL strain male.mice (each weighing
,¦22 + l g), each group consisting of lO mice, by using the
¦¦penicillin G-resistant strain of Staphylococcus aureus obtained
~in Example 1. The penicilli G-resistant strain of Staphylococcus
aureus which had been cultured in a Trypto-soy agar medium
¦(Nippon Eiyo Kagaku Co. Ltd.) at 37C for 18 hr. were suspended
¦in a Trypto-soy bouillon(Nippon Eiyo Kagaku Co.Ltd.) containing
15~ of mucin, and 0.25 mQ of such suspension was intraperitoneally
¦inoculated to each mouse. The inoculum was controlled to be 5 x 108
¦cells per mouse. Two hours after the inoculation penicillin G was
~intraperitoneally administered at the dose of 2.5 x 10 U/kg (body
,weight of mouse) and 5 x 104 U/kg, respectively, while the present
¦substance obtained according to the process of Example l was also
intraperitoneally administered at the dose of lO0 mg/kg (body weight
~of mouse). The thus treated mice were observed every day for the
period of 7 days after the administration, and the rate of survival
llof the inoculated mice was determined. The results are shown in
i~igure 3. It was proved by these results that the combined use of
¦ithe present substance, the nitrogen containing polysaccharide (NP),
can improve the remedial effect of penicillin G while elevating
~the sensitivity to penicillin G of the resistant bacteria in vivo.
jlEXAMPLE 3
25 ¦i A similar therapeutical test for infectious diseases was
carried out five groupe of ICR-~CL male mice ~eacb weighing 22+1 g,
I - 21 -
I, . 'I
'' -- ' ^`'-' :
110(~87~
an~ each gro~p consisting of 10 mice) by using the tetracycline-
resistant Escherichia coli and the present substance prepared
according to the procedure of Example 1. A quarter milliliter of
¦ a Trypto-soy bouillon (see Example 2) suspension, including 5%
¦ of mucin, of the tetracycline-resistant bacteria prepared as in
¦ Example 1 was intraperitoneally inoculated to each mouse. The
¦ inoculum was 5 x 108 cells per mouse. Two hours after the
¦ inoculation, tetracycline was administered orally at the doses
of 80 mg/kg (mouse body weight) and 160 mg/kg, respectively, with
simultaneous oral administration of the present substance at the
dose of 100 mg/kg. The results are shown in Figure 4 by way of
the rate of survival during the 7-day period after the adminis-
tration. It is apparent that the combined use of the present sub-
stance and tetracycline can produce a far higher remedial effect
than when tetracyline is used singly. Also, since much the same
¦ effect can be derived from oral administration of the present
¦ substance as from its intraperitoneal administration, the present
¦ substance can be typified by a broadened scope of use beside its
¦¦ high efficacy.
jj EXAMPLE 4
ll The penicillin G-resistant of Staphylococcus aureus were
¦1 inoculated at the rate of 5 x 108 cells/mouse to five groups
¦¦ (10 mice a group) of ICR-JCL male ~ice (22+1) according to the
~! procedure of Example 2, followed by intraperitoneal administration
of 2.5 x 10 D/kg of penioillin G and 1, 10, 100 and 1,000 mg/kg
- 22 -
il I
110~876
of the present substance, respectively to 4 groups (same as used
in Example 1). The daily observation of these mice for the
period of 7 days after the administration gave the results shown
in Fig. 5.
EX~LE 5
A similar therapeutical test Eor infectious diseases was
carried out using six groups of ICR-JCL male mice, each group
consisting of 10 mice inoculated with 5 x 100000000 cells of
tetracycline-resistant escherichia coli/mouse by using the
procedures in Example 3.
Two hours after the inoculation, tetracycline was
administered at the dose of 80mg/kg (body weight of mouse) with
simultaneous oral administration of the present substance used
in Example 1 at the respective doses of 0, 10, 100, 1,000 and
10,000 mg/kg, the sixth group being control. Observation of
the mice was daily carried out until the seventh day and the
results obtained during the observation are shown in Fig. 6.
EXA _ E 6
CM-151 strain of Coriolus hirsutus (Fr.) Quel (Ferm. Res.
20 Inst. Deposit No. FERM-P 2711j was cultured as in Example 1, and
then extracted and refined to obtain 17.5 g of powdery substance.
The properties of this substance, as determined according to the
methods of Example 1, were as follows:
(1) Elementary analysis : 43.7% of carbon, 6.4~ of hydrogen,
and 5.5% of nitrogen.
(2) Specific rotatroy power : [~]2D5 = ~30O
(3) Saccharic composition : 79% of glucose, 15% of mannose,
2~ of xylose, 4~ of galactose and
trace of fucose.
(4) Protein content : 33~
(5) Ratio of saccharide bonds (~ 71/29
- 23 -
l~OU876
(6) Average molecular weight : 95,000
t7) Composition of protein portion :
15~ of aspartic acid, 9% of threonine,
5~ of serine, 14% of glutamic acid
6% of proline, 9% of glycin, 9% of
alanine, 7% of valine, 1~ of methionine
5% of isoleucine, 6~ of leucine,
2~ of tryptophan, 4% of phenylalanine
2g of lysin, 3% of alginine, 2~ of
ammonia, 1% of N-glucosamine and each
trace of cystine, tyroshine and histi-
dine.
The following experiment was then carried out on the
present substance having the above-mentioned properties.
The chloramphenicol-resistant Salmonella enteritidis
were obtained as in Example 1.
A quarter milli-liter of suspension of the afore-mentioned
chloramphenicol-resistant bacteria in Trypto-soy-bouillon t$ee
Example 2) including 5~ of mucin was intraperitoneally inoculated
to each mouse of 5 groups of the mice, each group consisting of
10 mice. The inoculum was 1 x 10 cells/mouse. Twenty fi~e
hours after the inoculation, 50 mg/kg of chloramphenicol was
administered intraperitoneally. 500 mg/kg of the present sub-
stance was further given orally to another group. The rate of
survival 5 days after the administration was 40% in the group
where chloramphenicol alone was administered, while it was 80~
in the group where the antibiotic was given in combination with
the present substance.
ExAMpLE ?
3~ The streptomycin resistant ~lebsiella pneumoniae were
obtained by the procedure of Example 1 and 0.25 mQ of a Trypto-
- 24 -
110~876
soy bouillon (see Example 2) containing 5% of mucin and these
bacteria was intraperitoneally inoculated to each mouse of 2 groupC
of the mouse, the group consisting of 10 mice at the rate of
1 x 108 cells per mouse. Three hours after this inoculation,
100 mg/kg of streptomycin was intraperitoneally administered.
Another group was further given 120 mg/kg of the present substance,
obtained by the description in Example 6, via intraperitoneal
administration. The survival rate 6 days after administration
was 50% in the group where streptomycin was used singly, but
it was as high as 80~ in the group where the present substance
was used with streptomycin.
EXAMPLE 8
A quarter milli-liter of a suspension of the colistin-
resistant Pseudomonas aeruginosa cultured by the same procedure
in Example 1 in Trypto-soy bouillon (see Example 2) including
5% of mucin was intraperitoneally inoculated to each mouse of
2 groups of ICR-JCL male mouse tbody weight: 22+1 g, 10 mice
a group) at the rate of 1 x 108 cells/mouse. Two hours after
the inoculation, 190 mg/kg (l~g = 30 U) of colistin was administer~d
intraperitoneally. The present substance obtained by the same
procedure as in Example 6 was also orally administered at a dose
of 1,500 mg/kg. The rate of survival 6 days after the adminis-
tration was 40% in the single colistin group and 70% in the group
of joint use of the present substance.
- 25 -
