JPS5925330A - Antitumor agent - Google Patents

Abstract

PURPOSE:To provide a carcinostatic agent containing actinomycin S3 as an active component. CONSTITUTION:Actinomycin D and actinomycin S3 having antibacterial activity can be obtained from the cultured product of a bacterial strain KY 11634 (FERM-P No.4673). Actinomycins known as antibiotics include actinomycin D, C, C2, S3, etc. The actinomycin S3 (LD50: 0.35mg/kg, i.p.) having higher activity than the actinomycin D having antitumor activity is used as an active component for a carcinostatic agent. It is administered by intravenous injection, peritoneal infusion, etc. Preferable doses for intravenous injection or peritoneal infusion: 2-4mug/kg daily for continuous administration and 10-15mug/kg per dose for intermittent administration (once/7 days).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anticancer agent containing 1 actinomycin 83 as an active ingredient.

Actinomycin is known as an antibiotic, and examples include actinomycin C+02 and 83.

Among these, actinomycin D is known to have anti-pulmonary injury properties, but there is no such threat regarding actinomycin s8.

Unexploded lν1 Nagisa et al. 1/J. Regarding actinomycin s8, 4yth4+ 4jJi {'I4.1 was found to have b'l+1=1/1 activity compared to actinomycin D, and the present invention was completed. The microorganisms are Streptomyces antibioticus and Streptomyces chrysomalus. Streptomyces sp. I30P476.

Streptomyces frapus, Streptomyces frapus
It is known to use ``flame'' lass (C
ILC landbook of Microblo
logy vol II+, 1973').

The present inventors discovered that an antibacterial substance accumulates in the culture of a bacterial strain (referred to as KY11634) isolated from soil in the suburbs of Shimoitsukaichi-cho, Tokyo. They learned that it was Mycin S3 and that the strain was a microorganism belonging to the genus Strezomyces, and that it was a different strain from known bacterial species.

The above-mentioned Shida KY11634 has been deposited at the Institute of Imitation and Biological Technology, Agency of Industrial Science and Technology (its deposit numbers are Jinj, Weiku QII).
+ + ¥4 Yori No. 4673). Furthermore, the strain l'
, is also entrusted to NJ eyes LL (that snoring scent is,
N old also L] ] 395).

Next, regarding the mycological properties of +1ri+'l' (KY 11634), -rMVl', Akira I.

(A) Realistic results] (Y11634) successfully colonizes aerial mycelium on a general isolation medium, and its morphology is simply branched and spiral-shaped. The spores are 1 The surface is smooth.The shape of the spores is oval (0,4~
0.5 μ×0.7 to 0.8 μ).

Growth status when KYl16:i4 was grown on the ground, color and color of the front and back sides of the colony.
Table 1 shows the tTJ dye.

Color display is C, 'o1or 11anna11y Af
annual(ContainerCorporali+
This is according to the color classification by m of Anerica).

(13) Physiological Properties The physiological properties of KY//<74t are shown in Table 1.

Regarding things other than temperature, effects on milk and cellulose, we show the observation results after one week at 27℃, and the temperature! The results after Fi/month are shown for the effect on eye removal, milk and cellulose.

Physiological properties of NG, 2@KX//63 (1) Assimilable carbon source Assimilable D-arabinose 1 ~ D-xylose - D-glucose Sho D-fusictose (1, Sucrose II Inositol H- L-Rhamnose Raffinose D-Mannitol 11 (2) Liquefaction of gelatin None 3) Effect on milk Liquefaction Yes Peptoneization None 4) Decomposition of cellulose Slightly present (5) Hydrolysis of Seishin Yes (6) Optimum L to A l) 14 A
, r ~7. j (7) Optimal growth temperature, 21
~JI''C (3) Production of tyrosinase None 9) Production of melanoid pigments None Based on the above characteristics, KY//jJ4tlil!1 is classified as an actiform 1, which is 1 level above the streptomycetes.

Regarding this resistance, B, Ku8Cttr(
Illtθr11. J, 5yEl tent,
Bacteriol, , 2.2%/laJ.

According to the classification by p. 73, /7.2), this cocoon is classified as Stretomyces botroensis. However, W, B, Bhir:kingandkl
, fjoLtlleb (intsruatlamil
J, Systematici>act, arl
aloHy/'/ (41jri/, /ribu2) It has been shown that Stretmyces botroensis has a weak ability to assimilate raffinose.

In addition, the spiral state of the hyphae is a '71ζ shape with l-1 spirals, and it is an open type spiral state (open8
It is described as having fP (piral). However, gYiitJ≠ utilizes 2-fumefnose extremely well, on a par with glucose, etc., and is more densely packed than the helical type t, taking all forms of co+npac; aplra:l, opθn oplrai tJ, and unacceptable. Accordingly, KY//JJ# is clearly different from Strezotomyces botroensis.

゛On the other hand, Stregutomyces humpeolus is known as a producing bacterium of actinomycin S, but this bacterium is
r-u+ina L;1va Llac teriol
According to Ogy (J edition), the production of melanin is increased by IJ2<, ゛also BW child-Li numerous hairy bodies f;pj'L (iiiairly 8p(Ir
It is clearly distinguishable from KY//A344 in that it has an e-Lee C awn.

There! , Y//63≠ is assumed to be the sex fungus 4, and from the row of Streptoma f.
11aOell171J8ulj K Y11634
It was named 'l■ITTA).

In culturing this bacterium, the usual culture method of 1jl'itN is generally used. Various sources of nutrients can be used for culture. As carbon sources, glucose, starch, dextrin, mannose, fructose, sucrose, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, etc. Natural nitrogen sources include peptog, meat extract, yeast extract, dried yeast, corn, etc.
Steep liquor, soy flour, casamino acids, etc. are used alone or in combination. In addition, inorganic salts such as table salt, potassium chloride, calcium carbonate, and phosphates are added depending on the conditions, and organic substances and non-receivable substances that promote the growth of the bacteria used and the production of actinomycin J) and S3 are added as appropriate. Can be added.

As for the culture method, liquid culture method, especially deep agitation culture method is most suitable. The culture temperature is 25 to 40℃, especially 2
The optimum temperature is 8 to 38°C, and the medium should be rolled with ammonia water, ammonia carbonate water, etc.
~10, preferably 6-8! ? 'k.? It is desirable to do the same. When culture is usually carried out for 7 days in liquid culture, the target substance is produced and accumulated in the culture. Preferably, the culture is stopped when the production tail in the culture reaches the maximum. The target product is purified and isolated from the culture solution obtained by separating the bacterial cells and from the organic solvent extract of the bacterial cells.

Actinomycin 1) and S3 mono-I from culture medium
LLj' production utilizes separation and purification methods commonly used to isolate metabolic products of microorganisms from their culture fluids.

The present inventors (“Anti-1 for actinomycin S3
1Kt(jmu1sexualitykk'lpe)The following results were given.

(1) Therapeutic effect on sarcoma/rθ ascites-type tumor z x io' sarcoma/rθ ascites-type tumor cells were subcutaneously transplanted into the axillary region of four ddY male mice (approximately 201)/$lp. At hour V after transplantation, each patient was treated with cutinomycin S and phosphate buffered saline (1'B).
s) Solution 0. J m was administered intraperitoneally seven times. The composition of PB8 is 1JaCe O9J',! i'
/dl, KG/0. OJ, fi'/d
l, Ha2 HPOy/, /, t j//di,
Klil Miko 13o, 0.2 N/di, (p
kl Z co).

li as ratio 4'2 reli! After Ill tumor transfer 4i, mitomycin C (11-containing 0..2M) was administered intraperitoneally on the FB day. 4.4]<(mm and T/C(
T: average of test examples 1 hll 5 bodies 0 (, C: control example (
P Hkl O,, 2 ml administered intraperitoneally)) average +1! (l tx5 bodies) was measured, and the result was the same as the 31st one.

Silk 3 type agent administration style average jull tumor yellow 'l
'/U(+J?/As+) (+411')
ACNANOMER f/n” J O (against breakage)
/ ≠ , 20 -o, , 2z
,2ra o,,2゜θ,/,2!
4tt2 0. J'1 Might My/N C
: , rA J Tat
o,,xr(2) Lymphocytosis Lykemia P-3♂! Regarding the therapeutic effect on the lung field, 20 F/CIJ de/male mice/No 1 to 4, Lynholi Ideitik φ Rychemif' (Lyn le 1
toeytLceIjuke+oi, u) P-J I
r ji former gAIlfllu / x to
'l hard ・1 anti 111 (inscribed 4a. After transfer 4Vt 2
Acuna/-v-f Shin u, t, z) P at Vth hour
On day 13, 19 hL O, 2 hr6 was given to each litter of 18, 4 days. As the specific softness ν・1j, Ifl Aej t
real Jfu transfer pt>, Ma I at xy time]・
PB8gIIi O, -2+nt i I of Mycin C
11'P' administered IV 1111, r M2ke/
child. Average survival IN ik and I'/C (T
Table 4 shows the mean survival of the test samples: LJ k2, C: average survival of the control samples.

Table 4 Actinomyces 83 0 (control) 12.4
-0.125 19.6 1.5
80.0 (3220,01, (i27・r Tomycin C4,217,11,38-am) (3) Therapeutic effect on IJ-16 melanoma Body 1i approximately 20f ddy fiber 1 in 10 mice per group Ataυ13-
1 (10%0% homogenate of melanoma cells 5
mg was phase-shifted into the abdominal cavity. At 24 hours after transfer, phosphate buffered aJMIJt water (pBs) of medium strength actinomycin S3 or actinomycin D as a comparison was added.
> Solution Q, 2 ml, was intraperitoneally administered once every 24 hours for a total of 9 times.

Incidentally, the composition of the above PIIS is NaC6O, 8flit
.

KCI 0.02 flit %Na2Ill'O<
1.15 F/dl.

Kll, PO40,029/dl (p l(7,2
).

1' scale from 1 to 6o [T: average Ik &
! Tumor flt't/l, C: Control example (PH10, 2m/l)
! Intraperitoneal placement Iψ1. Table 5 shows the average tumor volume and cure rate of A).

Table 5 Actinomycin 8,200-1100
186 550 112
2 Akuwotsumai/n], + 1ooo
140 3500. 137
1250 143 31
25 128 (1 therefore '1
゛6, cure rate of 88 is better. Also, the amount of administration required for S3 is smaller. However, S3 has stronger IJ properties, but even taking that into consideration, S3 is better. In other words, for the maximum effect: J' and D were shown to be 1000μ2, but s3 is 100μ? Met.

Actinomycin 53 (LD6゜:035 revised qAy, i
, p, ) as an anti-pulmonary ulcer agent, it can be administered by intravenous administration, intraperitoneal administration, or the like. In this case, the daily dosage range is as follows: That is, in the case of continuous intravenous and intraperitoneal administration, it is about 2 to 4 μfAp/E3, and in the case of intermittent odor administration (1 [FiV 7 days), it is about 10 to 15μ? It is better to go with one Ap.

Next, an example of the production of actinomycin S3 will be shown.

Hano g 91J/.

[111 MJ and °C using Streptomyces meleochryseus NY//jj4'/l,. The strain is λl
4 ■ Medium in Erlenmeyer flask in valley E [Meat extract/11/d
1% fermentation extract/I/di, Nae/0.
Medium pH 7:2 (before sterilization) with a composition of 7 I/di)
Let's take a picture of the time (KoJQr, p, 01.) Culture-1
Il! L, ta. Thus obtained jt fill culture JO
Inoculate the fermentation medium irz of the lower ILi group J in the I-volume jar fer-mentor at a ratio of 30
℃, #air stirring method (rotation speed J j Q r, )),
Culture was carried out using ll1-r yingqibin/jl/n+in).

Fermentation medium composition Nigel course: 17N/l,
SJ A J)/l, L-asparagine 7, jllll
.

Kn, 2Po41o, /j//l, ~upper IP
L), 0. λ/I/l.

MgL3 (J, 7ooo, ori!/l, Yeast Exco 9/11 Bikumi y 11/ / (7s+y/l
, Biotia 30 μ&/l.

Fd IJU, ・711J I-1/ θ”rl
/ l s ”C12j Omy/ I!, Mn
1JtJ, -II, (l y ny / l,
'1nk304Iφ7) and uL(L
)1, jll, o Jh9/ 1opHA, I (
ffli%'1lil) to 14 a 01 (adjust.

Culture media J+! +p14f was cultured for 7.2 hours without control. Remove bacterial cells and precipitates from the culture solution, P7
I liked 戊／31.゛First, the active substance is adsorbed using nonionic porous I5 resin (trade name 1-up-, zOJ Mitsubishi Kasei 10) at a concentration of ≠o% acetone water (V/L) after washing with water. V) to remove impurities. Then J
'Elute with 77% acetone water (V/V). Concentrate the acetone fraction to dryness, dissolve in chloroform, and remove insoluble matter. This chloroform solution was combined with the extract from the bacterial cells described below and subjected to Norica gel chromatography.

Suspend the bacterial cells in acetone and extract Purpose C (Nagga). The amount of acetone used is about 100g. Dissolve the extracted lα in chloroporum and remove impurities. The chloroform solution obtained here was combined with the chloroform bath solution previously diluted with the culture medium F71 (approx.
), solica gel (trade name: 8111c ARCC-p Malli) filled with chloroform as a bath medium in advance
nkrodt Co, 1), E! , yeah )
(j Qo 11 ()), washed thoroughly with chloroform (approx. Kokinmono is served in a bath.

1 live music 1) was converted to formula 0 (d) to obtain crude standard/g.

It is a mixture containing cutinomycin 3 and acnanomycin D in a ratio of 72 J. [How to obtain both components from the standard sample?] This was done using the JHA Paper Claw 71 method. The decoy bath items are 1r -bu shiy1θti1θl
: terashi rachloro eshi1tIIIne
: / 0% sodium crout,
1 natu (J:/:J) (V/V), and the paper used was Toyo υA paper paper jθ. After development, take all the corresponding two parts, Aceto/de? Valley out/ζ. Prostitute J(if
After drying and crystallizing from the basil enal, Acanomyces cerevisiae/8. θ−t I , Achnanamine 7 Dθ. I got iry.

The physicochemical properties of acnanomycin 8J and D, which were thus determined, were as follows, and were similar to those of the 7J [(') inhibitor.

°f cutinomycin 8J th point, 24t〆~, 2gu a"c (decomposition) Shiji: A11) 41 minutes (1: C:
Jt, 4tO rice, kJ:i 3. it chi (yoke 1
(Direct) 111. jtchi UV absorption etc. p4z, 4111u% 2I/LOn
II] Amino (Subun 4 'I' b r, /,
, 2/%k, P r o, /, 00 mol, Ox
o Pro, 0.27 mol, Va/.

, ZOII mol, Sat, /, rit mol, N-Me
-vnL JLOj molar cooking (1'1ll- is known that 7 parts of hydrolyzed water is destroyed in t, and it is originally 2 mol, but in actual analysis, the data will be the same as above).

[α]J(/-i Jza110 (c)1eJ,
C=7.0) Acnanamine 7 D HI 3 points, 2 degrees θ ~, 2 degrees Celsius (zuHyl) no elementary analysis 1 + ri C: r '1. zs%,
IIAJ Za96, (Central 61 old) 1 (: / 3.
4Z 3≠Amino acid content 1r'l゛klr, /, JII
Mol, Val. , ZOa mol, Pro 100 mol, 8ar, /, P 1 mol, 11-Me-va110
3 moles of nail (Tlxr is known to be destroyed in 7 parts during hydrolysis and is originally 1 λ mole, but the actual analytical data is consistent with the above).

What is interesting is that the bacteria used in the present invention are overwhelmingly prolific in producing actenomycin.

Consideration Example 1: Create a persimmon mother in exactly the same manner as Example 1, and follow the culture method.
The culture conditions and culture time were also exactly the same as in the case study.

However, the fermentation medium used had the following composition.

Fermentation medium composition = 7 euculose aog7t, glucose/OJ//1. Fructose 1017t% Corn Steep Liquor” l/ts Nacl J I/
l s Me day "u・7" JO/i/l, HaPo
y 21//Is (tau,), so.

1rti7z, vitamin B/""9/7s bio-7-
7JOILl//l,)I'eBO,・711. ,()
1089/l, CaC1,2JOr+971,
MnBO, -11, U 4L'a9/1%Zn5O,・
711,20 j 019/l.

Cu5O,I JH20J ``9/ j o ];'
1N&-J' (before sterilization) Adjust with) J a O) i.

Actinomycin was produced in exactly the same manner as $4=4/ to produce actinomycin S.

o, r, p 1 actinomycin DO, 2 g was obtained.

Claims (1)

[Claims] An anticancer drug containing actinomycin S8 as an active ingredient.