FR2569983A1 - SPF-140 TUMORICIDE SUBSTANCE AND PROCESS FOR PREPARING THE SAME - Google Patents

Abstract

NEW TUMORICIDE PRODUCT, CALLED SPF-140, OBTAINED FROM STREPTOCOCCAL CULTURE. IT IS PREPARED BY ADDING TO THE CULTURE MEDIUM, AT A SUITABLE TIME, PENICILLIN, OR ONE OF ITS DERIVATIVES, SO AS TO RELEASE THE ACTIVE SUBSTANCE FROM STREPTOCOCCAL ORGANISMS IN THE CULTURE BROTH; THIS IS THEN FILTERED AND THE FILTRATE IS ADDITIONED WITH AMMONIUM SULPHATE; THE RELEASE PRODUCT, CORRESPONDING TO SATURATION DEGREES OF 50 TO 90, IS DISSOLVED IN A BUFFER SOLUTION WHICH IS THEN SUBJECTED TO TREATMENT BY ION EXCHANGE AND GEL FILTRATION CHROMATOGRAPHY. A RELATIVELY PURE PRODUCT IS OBTAINED, WITH A MOLECULAR WEIGHT OF BETWEEN 25,000 AND 70,000.

Description

The present invention relates to a novel

  tumoricidal substance, SPF-140, and to a method for

parry.

  It has already been used in carcino-

  static, living Streptococcus pyogenes, attenuated. On the other hand, are already known: a process which consists in

  grind the bodies of S. pyogenes, to extract one

  killing active with water or saline, which is

  then add an organic solvent, in order to collect

  lir, as a precipitate, the tumoricidal component

  (See Japanese Patent Publication No. 1647/1963); another

  a process which consists of a bacteriolysis of S. pyogenes using a lysokinase such as lysozyme or cellulase, or a proteinase, and fractionation of the active fraction in the form of an aqueous phase (Cf English patent

  N 1163865); and a method of grinding

  pyogenes, to collect the

  in water, and to treat them with nuclease and

  proteinase (Japanese Patent Laid-Open No. 7014/1980).

  As indicated above, it is well known that the streptococci themselves, or some of their constituents, exhibit tumoricidal activity. However, the substances usually known are mainly bacterial cells or cellular constituents,

  polymers, soluble or insoluble. In order to isolate a

  killing of a bacterium it is necessary to carry out

  bacteriolysis or mechanical grinding of the bacterium

  and split the whole. These different

  may complicate the purification of the product,

  isolation of the active constituent is very difficult.

  cult. The only current example of an active ingredient having been isolated relates to a molecular weight protein 000 (see Japanese Patent Publication No. 43841/1973

  and Japanese Patent Laid-Open No. 44617/1976).

  The Applicant has previously carried out studies to isolate an active tumoricidal component, produced by streptococci, and succeeded in isolating the new physiologically active substances SPF-1 and SPF-2, as well as a new tumoricidal substance SPF-100, by addition. penicillin, or derived substances, at a suitable time during the cultivation of streptococci, further cultivation and recovery of the substances mentioned above, present in the medium, from the broth

  filtered culture, followed by purification. These substances

  these inhibit the growth of a mutant permeable to macro-

  molecules of an MP-2 colon bacillus (FERM-P5432) (See Agr.

  Biol. Chem. 43, 371-378 (1979) which will be referred to hereinafter

as a strain of MP-2.

  The present invention is based on the observation

  The filtered Streptococcus broth contains, in addition to SPF-1, SPF-2 or SPF-100, a fraction that does not inhibit the growth of the XMP-2 strain, but

  has a tumoricidal effect. The substance of this fraction

is called SPF-140.

  The new tumoricidal substance SPF-140, according to the invention, obtained in the filtered Streptococcus culture broth, treated at a suitable time with penicillin or its derivatives, is characterized by a weight

  from 25 000 to 70 000 and in that it does not

  not hibe the growth of the strain of MP-2.

  No known tumoricidal substances are known

  S. pyogenes, secreted and accumulated in a

  medium, and having a molecular weight of less than

  zaines of a thousand. In addition, the ultraviolet absorption spectrum of the substance SP-140 according to the invention, which appears to be of peptide nature according to the results of the elemental analysis and the color reactions, suggests

  that it is a new substance different from the sub-

known tumoricidal stances.

  The present invention includes a method for

  preparation of this tumoricidal substance SPF-140, which

  takes the culture of an SPF-140 producing streptococcus and the recovery of the tumoricidal substance from the medium. The invention is described in more detail with reference to the accompanying drawings, in which:

  FIG. 1 represents an absorption spectrum in the ultraviolet

  violet of a 0.1% aqueous solution of SPF-140, while

  FIG. 2 represents an absorption spectrum in the infra-

red of the same solution.

  During the process according to the invention, any species of streptococcus capable of producing the tumoricidal substance SPF-140 can be used. In particular, Streptococcus pyogenes ATCC 21060 Streptococcus sp. ATCC 21597 Streptococcus pyogenes ATCC 21546 Streptococcus pyogenes ATCC 21547 and

Streptococcus pyogenes ATCC 21548.

  Natural media are frequently used as meat extract medium, yeast extract medium, and heart-brain infusion medium (BHI medium), although any medium suitable for growth of streptococci may be used. The streptococci can be cultured under appropriate conditions, i.e. at a pH of 5 to 8 (preferably 6.1 to 7.2) and at a temperature of 30 to 40 ° C (preferably 35 to 37 C). In general, we use

  an anaerobic and static culture, or with agitation.

  During the process according to the invention, the provision of penicillin, or substances derived at a suitable time, plays a large role in the recovery of the tumoricidal substance SPF-140,

  Penicillin, or the substances derived from it, may

  to be added 3 to 20 hours, and preferably 5 to 10

  hours after the beginning of the logarithmic growth phase.

  that when the bacterium is cultured at 37 C. The continuation of the culture for 1 to 20 hours, and preferably 3 to

  hours, should allow the accumulation of a large

  SPF-140 tumoricidal substance in the medium.

  It is possible to operate with any substance derived from penicillin, known to have a similar effect, although penicillin G is usually used. It is added in a proportion of 100 to 7000 units / ml, preferably

300 to 5000 units / ml of medium.

  The culture broth thus obtained is centrifuged

  to remove cells, and am sulfate is added.

  monium to the filtrate. After recovery of the corresponding fractions

  50% to 90% saturation levels, the release product obtained containing the tumoricidal substance

  SPF-140, may be stored in frozen form.

  The substance SPF-140 is purified from the salting product by dissolving the latter in a buffer solution, followed by adsorption and elution of the solution thus formed, continuously or not,

  with an ion exchanger, followed by a chromatography of

gel treatment.

  Ion exchangers are suitable as ion exchange resin, ion exchange cellulose, ion exchange Sephadex (a product of Pharmacia AB) or hydroxylapatite,

  and for gel filtration, substances such as Toyo

  pearl HW 50F or HW 50SF (products of Toyo Soda Mfg. Co., Ltd.) or Sepha4ex (product of Pharmacia AB). Adsorption of the tumoricidal substance SPF140 by the exchanger. ion,

  and its elution from this substance, may be

  kill with a buffer solution of suitable pH value and salt concentration. We can possibly

  combine two or more ion exchangers or

these gel filtration.

  The tumoricidal substance SPF-140 in lyophilic form

  philized obtained in Example 1 as described below,

  is a pale yellow powder having the physical properties

following chemicals.

1. Basic analysis

C: 42.58 to 41.79%

H: 6.15 to 5.92%

N: 12.69 to 12.35%

O: 36.33 to 38.78% and

ash: 2.25 to 1.16%.

2. Molecular weight;

  Approximately 25,000 to 70,000, as determined by

mined by gel filtration.

  3, Decomposition Point: The product browns at 170 ° C and darkens at 245 ° C, and decomposes, 4. Specific rotatory power:

D20 = -70 to -80 (C = 1.0).

  D 5. Absorption Spectrum in the Ultraviolet: Figure 1 shows an absorption spectrum in the ultraviolet of a 0.1% aqueous solution of SPF-140, which is characterized by the maximum absorbance at 275 nm, 6. Absorption spectrum in the infrared: Figure 2 shows an absorption spectrum

  in the infrared of the same solution.

  7. Solubility in solvents; The product is soluble in water, partially soluble in methanol, and insoluble in solvents including ethylene, n-propanol, n-butanol, isobutanol, n-hexane, chloroform, acetone, methyl

  isobutyl ketone and diethyl ether.

  8. Acidity: The pH value of a 1% aqueous solution is

of 6.5.

9. Form:

Pale yellow powder.

  10. Color Reactions Ninhydrin reaction; positive Biuret reaction: positive Lowry reaction: positive Molisch reaction: negative Dische reaction: negative Anthrone reaction; negative, and

  Reaction with cysteine sulphate: negative.

11. Stability:

I do not require stabilizer.

  In the present invention, antelab

  is determined by the growth inhibition effect of strain MP-2. In addition, the unit of activity of a physiologically active substance is determined by the method of Udaka (see J. Antibiotics, 35, 1319 to 1325 (1982)) with indication of the antibacterial activity against MP-2. That is, medium (M3 medium) comprising 1.75% "Bact Antibiotic medium 3" (a product of Difco) and 1.3% agar, is sterilized by heating at 120 ° C. for minutes. Then 20 ml portions of this medium are poured into petri dishes, and allowed to cool.

  to prepare the plate media.

  On the other hand, a medium which comprises 0.5% of peptone is sterilized by heating at 120 ° C. for 15 minutes.

  0.5% meat extract, 0.3% NaCl and 0.8% agar.

  Then we put it in a thermostat at 420C. When the temperature

  this environment reaches 420C, PM-2 is added

  grown at 370C for 17 hours at a rate of 104 cells / ml. 2 ml of this medium are collected using a pipette and deposited on the surface of M3 medium which has been previously prepared. Then the added medium is

  immediately spread homogeneously, and is solidified.

  A test solution is suitably diluted. A paper washer (diameter 8 mm, a product of Toyo Roshi Co. LTD.) Is impregnated with this solution, and is then placed on the plate prepared above, and the

  culture is conducted for 17 hours at 37 C in order to determine

  to decrease the diameter of the zone of inhibition resulting from the test substance. Then we define as correspondent

  unit (1 u) the concentration of the substance of es-

  sai for which the diameter of the zone of inhibition is

10 mm.

  The present invention is illustrated by the

following non-limiting examples.

EXAMPLE 1

  1 ml of BHI medium are inoculated with Streptococcus pyogenes ATCC 21060 and subjected to static culture at 37 ° C. for 8 hours to obtain a culture fluid.

  of germ. Then one sows with this fluid 1 liter of half

  Place 1, having the composition indicated in Table 1 below, and subjected to an anaerobic preculture under conditions identical to those observed for

the culture of the germ.

  TABLE 1: Medium A Meat extract 1% Polypeptone 1% Maltose 0.5% Yeast extract 0.25% Monopotassium phosphate 0.1% Magnesium sulphate 0.05% (pH = 6.8) Then 8 liter of medium A are introduced into a fermentation flask (10 liters), are sterilized at 120 C during

  10 minutes, and allowed to cool to 37 ° C before sowing.

  1 liter of precultivated fluid; and one cultivates in ana

  robie at 37 C and pH 6.5 for 15 hours 30 at the speed of

  300 rpm. 5000 units / ml of

  nicillin G in this environment and the culture is continued

  5 additional hours. The culture broth obtains

  naked is centrifuged to remove the cells.

  Ammonium sulfate is added to the broth

  and recover a fraction corresponding to a degree of saturation of 50 to 90%, to obtain a precipitate. This

  precipitate contains 381 x 104u of a physiological substance

  active, which inhibits the growth of MP-2. It is dissolved in 300 ml of a phosphate buffer solution (1 × 10 -2 M, pH = 7, KH 2 PO 4 -Na 2 HPO 4) and the solution

  obtained is introduced into a DEAE-cell column

  lose (5 x 70 cm) so that the physiologically active substance is adsorbed therein. Then we gradually elect with

  the previously mentioned phosphate buffer solution,

  closing 0.3M sodium chloride to obtain a fraction

  having a physiological activity of 283.4 x 104 u.

  This fraction is adsorbed on a DEAE Sepha-

  dex A-25 (2.6 x 70 cm), and then eluted while increasing

  linearly the concentration of sodium chloride in the phosphate buffer solution mentioned above, to collect a fraction having a physiological activity of

  249.6 X 104u. The eluate is again concentrated and is introduced

  in a column (2.6 x 100 cm) of substance for filtration.

  on Toyopearl HW 50F gel, in order to collect a non-bactericidal fraction which after freeze-drying gives

  640 mg of the tumoricidal substance SPF-140.

  This substance thus obtained is used in test examples 1 and 2 described below to examine

its tumoricidal action.

EXAMPLE 2

  The Streptococcus pyogenes ATCC 21060 is cultured in the manner described in Example 1 and in medium B, the composition of which is indicated in Table 2.

  penicillin G at a concentration of 1000 units / ml of

  place. The filtered culture broth is purified as

  described in Example 1, and gives 767 mg of the

ricide SPF-140.

  TABLE 2; Medium B Meat Extract 0.5% Polypeptone 1% Yeast Extract 0.25% Sodium Chloride 0.1% (pH = 6.7)

EXAMPLE 3

  Streptococcus pyogenes ATCC 21060 is grown in the manner

  described in Example 1, in a medium C the composition of which

  The filtered culture broth is purified as described in Example 1, and gives

  527 mg of the tumoricidal substance SPF-140.

  TABLE 3: Medium C Meat Extract 1% Polypeptone 1% Maltose 0.5% Yeast Extract 0.25% Monopotassium Phosphate 0.1% Magnesium Sulphate 0.05% Sodium Chloride 0.5% (pR 6, 5)

EXAMPLE 4

  The culture described in Example 1 is carried out in medium D, the composition of which is shown in Table 4.

  Streptococcus pyogenes ATCC 21060. Adding penicillin

  line G until a concentration of 1000 units / ml of medium is obtained. The filtered culture broth is purified

  as in Example 1, and gives 830 mg of tumor substance

ricide SPF-140.

  TABLE 4: Medium D Maltose 0.8% Polypeptone 0.1% Yeast Extract 0.1% Monopotassium Phosphate 1.43% Magnesium Sulphate 0.585% Potassium Nitrate - 1% (pH = 6.5)

EXAMPLE 5

  Streptococcus pyogenes ATCC 21060 is cultured in the manner described in Example 1 in medium E, the composition of which is shown in Table 5. Penicillin is added

  G to have a concentration of 1000 units / ml of medium.

  The filtered culture broth is purified as described in

  Example 1, and gives 570 mg of tumoricidal substance SPF-

  140. TABLE 5: Medium E Maltose 0.3% Casamino acids 0.5% Yeast extract 1% Monopotassium phosphate 0.5% Magnesium sulphate 0.1% Sodium chloride 0.1% Potassium nitrate 0, 5% (pH 6.5)

EXAMPLE OF TEST 1

  The tumoricidal activity of the in vitro test agent is

  determined in accordance with the determination of the degree of

cell bition.

  L-5178Y (Leukemia), used as tumor cells, is suspended in RPMX 1640 medium containing 10% FCS and 5 mg / liter of kanamyclne. 0.45 ml of this medium is injected into a Falcon 2058 tube so as to have a concentration of 1 × 10 5 cells / tube. Then, a certain amount of the test agent, which is prepared by dissolving the tumoricidal substance SPF-140 in 0.05 ml of medium, is injected into the medium, and cultivated

  at 37 ° C in the presence of 5% C02. 48 hours after the addi-

  From the test agent, vital staining is performed with trypan blue, and the degree of cell inhibition is determined by the following equation. 1 1 Number of cells in each test group (A) inhibition = x 100 cell (% 1 Number of cells in the control group (A ')

  Table 6 shows the result obtained when using

  SPF-140 as the test agent.

  TABLE 6: Degree of cell inhibition (%) SPF-140 (mg / ml) L-5178Y

2.0 15.3%

1.0 11.3%

EXAMPLE OF TEST 2

  The tumoricidal activity of the test agent in vivo is determined using female CRJ-CD-1 ICR mice,

5 weeks old.

  Cancer cells, which are cells of ascites

  Sarcoma-180, are suspended in physiological serum

  siologique. Each mouse receives by intraperitoneal

  0.2 ml suspension containing 1 x 106 cells can-

céreuses.

  After this inoculation, it is administered intraperitoneally

  a specific quantity of the test agent, at each

  than mice, once a day for 7 days, in order to ob-

  serve the number of live mice.

  Table 7 shows the results of a test run during the

  the test substance was used as the test substance

ricide SPF-140.

  TABLE 7: Tumor Activity of SPF-140 (Number of Live Mice) Dose (mg) Number of Days

0 10 15 20 25 30

Witness 5/5 2/5 1/5 0/5 0/5 0/5

0.3 5/5 5/5 5/5 4/5 4/5 3/5

  The rate of prolongation of life (mean survival time in control group days / mean survival time in

  test group 10) days of the test agent is 270%.

Claims (10)

claims   1. Tumoricidal substance, called SPF-140, characteristic of   the following physicochemical properties: (1) Elemental analysis C: 42.58 to 41.79% H: 6.15 to 5.92% N: 12.69 to 12.35% O: 36.33 to 38.78% and Ashes: 2.25 to 1.16%.   (2) Molecular weight About 25,000 to 70,000, according to the determination by gel filtration.   (3) Decomposition point The substance brines at 170 ° C and blacks at 245 ° C. and breaks down.   (4) Specific rotatory power [y20 = -70O to -80 (C = 1) (5) Ultraviolet Absorption Spectrum Figure 1 shows an ultraviolet absorption spectrum of a 0 aqueous solution , 1%, which is characterized by a maximum of absorption at 275 nm.   (6) Absorption spectrum in the infrared Figure 2 shows an absorption spectrum   in the infrared of the same solution.   (7) Solubility in solvents The substance is soluble in water, partially soluble in methanol, and with difficulty, or not   soluble in solvents comprising ethanol   n, n-propanol, n-butanol, isobutanol, n-hexane, chloroform, acetone, methyl isobutyl ketone and ethyl ether.   (8) Acidity The pH value of a 1% aqueous solution is of 6.5. (9) Presentation Pale yellow powder.   (10) Color reactions Ninhydrin reaction; positive Biuret reaction: positive Lowry reaction: positive Molisch reaction: negative Dische reaction; negative Reaction to anthrone: negative   Reaction with cystine sulfate; negative. (11) Stability   The substance does not require stabilizers.   2. Process for producing SPF-140, according to the   cation 1, from a culture of Streptococci, characterized   in that the substance is released into the culture broth by addition of penicillin, or by-products, at an appropriate time during the cultivation. 3. Method according to claim 2, characterized in that streptococci, ATCC 21060, Streptococcus pp, ATCC 21597, Streptococcus pyogenes ATCC 21546, Streptococcus pyogenes ATCC 21547 or Streptococcus pyogenes ATCC 21547 or Streptococcus pyogenes are used as streptococci. Streptococcus pyogenes ATCC 21548.   4. Method according to one of claims 2 or 3,   characterized in that it is used for growing media   as a medium of meat extract, extract of   vure, or heart-brain infusion (BsI medium).   5. Method according to one of claims 2 to 4,   in that it is carried out at a pH of 5 to 8, preferably 6.1 to 7.2 and at a temperature of 30 to 40 ° C, preferably between 35 and 37 C.   6. Method according to one of claims 2 to 5, characterized   in that it operates in anaerobic, sta- that or with agitation.   7. Method according to one of claims 2 to 6,   tered in that penicillin G is added at 7000 units / ml medium, preferably at the rate of 300 to 5000 u / ml.   8. Method according to one of claims 2 to 7,   in that the penicillin, or a derivative thereof, is added, during a culture at 37 C, 3 to 20 hours, and preferably 5 to 10 hours, after the beginning of the phase of   logarithmic growth, and in that after this addi-   tion, the culture is continued for 1 to 20 hours, and   preferably for 3 to 15 hours.   9. Process according to one of claims 2 to 8,   in that the culture broth, after treatment   penicillin is centrifuged to remove bac-   before being added with ammonium sulphate.   10.Procédé according to claim 9, characterized in that the precipitates obtens, corresponding to degrees   saturation levels of 50 to 90% of ammonium sulphate are   dissolved in a buffer solution for treatment   by ion exchange and filtration chromatography.   gel to obtain the purified SPF-140 substance fied.