JPS62249927A - Proteinous substance and antitumor agent - Google Patents

Abstract

NEW MATERIAL:A proteinous substance having a main component molecular weight of 15,000-20,000 and obtained by physically disintegrating toxoplasma, subjecting the resultant water-soluble component to ultra-high-speed centrifugal separation and separating from the supernatant liquid. USE:An antitumor agent exhibiting remarkable antitumor activity even against allogenic tumor, isogenic tumor and autologous tumor. A synergistic effect can be achieved by the combined use with an immuno-activating substance ovioactin. It is easily soluble in water in contrast with an IFN-inducing agent disclosed in Japanese Patent Applitation No.59-27832. PREPARATION:Toxoplasma (e.g. Toxoplasma gondii) is diluted with distilled water and disintegrated by ultrasonic treatment at 4-15 deg.C for 3-5min. The insoluble components in the disintegrated product is optionally removed by centrifugal separation (e.g. at 16,000 G and 4 deg.C for 60min) and the product is converted to isotonic solution with NaCl and subjected to ultra-high-speed centrifugation at >=100,000 G (e.g. at 4 deg.C for 120min). The objective proteinous substance containing a main component having a molecular weight of 15,000-20,000 can be obtained as the supernatant liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a novel protein substance and an antitumor agent containing the protein substance as an active ingredient.

[Background of the Invention The present inventors first created a movement that induces interferon (hereinafter abbreviated as IFN) in the body of a cow, obtained by physically crushing Toxoplasma gondii. However, based on this finding, we filed a patent application (Japanese Patent Laid-Open No. 59-27832) regarding an IFN inducer.

That is, in the above-mentioned publication, Toxoplasma gondii (hereinafter abbreviated as TD) is crushed by a conventional ultrasonic disruption method, freeze-drying method, etc., and then centrifuged (10,0
The precipitate (vehicle body wall skeleton fraction) obtained by 0Orl) m or more, about 4 ° C) and the upper end were further subjected to molecular weight fractionation by conventional gel filtration method, membrane filtration method, etc. Water soluble fraction of 100,000 or more, molecular weight of 20
．． The present invention has disclosed an invention relating to a low toxicity, in vivo administrable IFN inducer selected from the water-soluble fractions with a molecular weight of 4,000 to 100,000 and the water-soluble 111 fractions with a molecular weight of 4,000 or less.

The present inventors conducted intensive research to purify and isolate the bovine immediately active l'l substance in Tr) water-soluble components. The present invention was completed based on the discovery that protein substances with a narrow target have excellent antitumor effects.

[Structure of the Invention] That is, the present invention centrifuges a water-soluble component obtained by physically crushing Toxoplasma gondii at 100,000 G or more to obtain an upper end, which has a main component molecular weight of 15,000 to 20.
,000 protein substances and antitumor agents containing the protein substances as active ingredients.

[Manufacturing method] As a raw material for producing the protein substance according to the present invention, any T
I) strains can also be used; a representative example is Toxoplasma bombii.
gondii). TF) that is commonly available may be used as it is, or it may be isolated and used after being grown in an animal body or in tissue culture cells according to a conventional method.

The physical crushing of TI) is carried out according to the conventional method as described in the above-mentioned publication using the ultrasonic crushing method and the freeze-thaw method alone or in combination? ``I can do something.

That is, when using the ultrasonic disruption method, TU) is mixed with distilled water, physiological saline, phosphate buffered saline (PBS), etc.
), a suitable buffer such as salt-balanced Hank's solution (TolB55), preferably distilled water, is usually used at 1001n9/m (
1 (2X 109 fine soil p/mQ) A solution or diluted solution is prepared, and this is treated with ultrasonic waves for about 3 to 5 minutes with a warm stone at 4 to 15°C.Also, when using the freeze-thaw method (Tr adjusted in the same manner as above)
The containing liquid was frozen in advance at -20'C to -80°C,
Next, the frozen product is thawed several times at approximately 20 to 37°C (
A desired crushed product can be obtained by repeating the process (usually about 5 times).

Next, TI) Insoluble matter in the crushed material is removed by centrifugation (e.g., 16,000 G, 60 minutes, 4°C), etc., if desired, and sodium Jn chloride solution, etc. is added to prevent denaturation. After straining, ultra-high-speed centrifugation at over 100,000G (
For example, by carrying out the reaction for 120 minutes at 4°C, the protein substance of the present invention having a main component molecular weight of 15,000 to 20,000 can be obtained in a solution state. This protein substance solution is sterilized by filtration, and if adjusted to an appropriate concentration, it can be used as it is as an antitumor agent, which will be described later. Powdered protein substances obtained by isolation by vacuum freeze-drying etc. can maintain their activity for a long period of time by storing them in a cool and dark place.

[Relationship between the protein substance of the present invention and the IFN inducer] Both the protein substance of the present invention and the IFN inducer disclosed in JP-A-59-27832 are protein substances obtained by physically crushing Tp. It is.

However, the protein substance of the present invention is a water-soluble component whose main component molecular weight is 15,000 to 20,000 and is obtained as the upper end by high-speed centrifugation of the water-soluble component of the crushed product. The IFN inducer of 1983-27832 was obtained by TI) centrifugation of crushed material (10,OOOrl)m
above, 4°C) The precipitate and the upper end were further subjected to molecular weight fractionation using a conventional method.
0~100.000. and 4,000 or less in that each is a poorly soluble or relatively poorly soluble component.

Therefore, the protein substance of the present invention is considered to be a new substance different from the protein substance disclosed in the above-mentioned publication.

Furthermore, although the above-mentioned publication describes the effect of inducing IFN@, there is no specific description of the antitumor effect.

[Anti-tumor effect] Tests on mice and rats revealed that the protein substance of the present invention has a significant anti-tumor effect against allogeneic tumors, syngeneic tumors and autologous tumors.

In addition, the immunostimulant substance opioactin (Obi oactin)
i n, see U.S. Pat. No. 4,482,543).
A synergistic effect was observed for Jf.

(1) Effect on allogeneic tumors (i) 3 arcoma180 (S-
180 > Cells I Every week, the protein substance of the present invention (Toxoplasma lytic antigen, hereinafter referred to as T.
It is abbreviated as IA. ) 100fJ'J to light mineral oil (I
MO) 0.2 m (!) for intramuscular use as an emulsion, and for third snoring, use Obioakboon 40 my/Ky in weeks 1, 2, and 4 [1st and 5th week]. TIA emulsion was administered intramuscularly in the same manner as in the second group.The tumor area (long axis I\1Ill x short axis mm'') of these three groups of mice was measured over time, and as shown in Figure 1, the control group ( Compared to a), the TI-Δ alone group ([)> showed a remarkable growth-inhibiting effect, and 7) the survival rate of [1] was 100%, compared to 40% in the control group.

Moreover, the third group (C) showed a more remarkable effect than the second group, and it was observed that it was effective in fixing the engraftment of tumor cells.

(ii) ICR/JCI-strain 4-5 weeks old 7. (Tumor-bearing mice were prepared by transplanting S~1ε30 cells into 10 mice in the same manner as in (i), and 5 mice were divided into two groups. The first group was used as a control, and the For group 2, Tl-A was administered weekly from the 1st week after S-180 cell transplantation as in the second group of (i).
100 μU of IMO (0.2me) emulsion was administered intramuscularly, and the tumor area was measured over time. Figure 2 (a) (control group)
, (b) (TLA administration group).

In young mice aged 4 to 5 weeks, administration of TLA alone was found to have a significant effect on stopping the intake of beef.

(2) Effect on syngeneic tumors 20 BAI B/C 4-week-old male mice were divided into 3 groups, the 1st group was used as a control, and the 2nd and 33rd groups were
00 u of 1-Mo (0,1 mN) emulsion was administered twice at 2-week intervals, by intramuscular administration in the right thigh for the second group and intraperitoneally for the third group.

4 weeks after the first TIA administration, syngeneic mice were given me↑hy
lcholan1. A suspension of 0.2 m (!) containing 5×10 MC-cells/ml of hrene (Me)-induced tumor cells was implanted subcutaneously on the back of a mouse, and the tumor area and survival rate were measured. Figure 3 (a)
(control group), (b) (thigh intramuscular administration BY), (C)
The results shown in (intraperitoneal administration group) were obtained. In other words, suppression of tumor cell proliferation was observed in the second and third groups from around 3 weeks after tumor cell transplantation, and this effect was more pronounced in the intramuscularly administered group (group 2), and the survival rate was On the 49th day after MC-tumor cell transplantation, the survival rate was 20% in the first group, but 80% in the second and third groups, and a significant survival effect was observed in the TIA administration group.

3) Effect on autologous tumors (i) 10 Wistar male rats (15
A paraffin pellet containing 5 mg of MCO was inserted subcutaneously into the back of a body weighing 0 to 200 g using a trocar, and divided into 2 groups of 5 animals.The first group was used as a control, and the second group was T L A O, 5Ir every month afterwards
e/body was subcutaneously administered as a saline solution into the first shoe or lower leg thigh, kept for 4 to 6 months, and the state of tumor induction was observed. As a result, as shown in Fig. 4(a), tumors developed in all rats (100%) at 21[] days after tumor onset on the 14th day. In the TLA administration group, the tumor incidence was 40%, indicating an inhibitory effect on tumor induction.

(ii) Similar to (i), paraffin pellets containing MC
Among the rats in which tumors were induced by inserting ~, 10 rats with liver tumor diameter of 1 cm or less (2 x 3 mm>) were selected, and these were divided into 2 groups of 5 rats, with the first group serving as a control. ,
1 LA to 0.5#lL' every week for the second group
The tumor area was measured over time. As a result, as shown in Figure 4(b), compared to the control group, TL△
A remarkable tumor growth suppressive effect was observed in the treated group. In particular, for two animals (40%), tumor growth almost stopped.

[Acute toxicity] Litchfield and Will] Tuxon (litsc
hfield & Wilcoxon) h method (J,
Pham, & Exp, Therapeutics,
90.90.1949), ICR/JCL adult 209 female mice, 3 in each group, were each given TI A20.
,50,100.200.50011,000110,
When 000150.000/1 g was dissolved in 1 d of physiological saline and administered intraperitoneally, no deaths were observed within 24 hours after administration.

Therefore, the LD50 value is 2500 mFJ for intraperitoneal administration.
/KFJ or higher, and it became clear that the toxicity of the protein substance of the present invention is extremely low.

[Application to antitumor agent] The protein substance of the present invention can be used as an aqueous solution or a physiological saline solution for the purpose of inhibiting metastasis and engraftment of tumor cells, usually in type A tumor cells.

In addition, if necessary, commonly used azicobant and various additives, such as solubilizing agents, emulsifying agents such as light mineral oil, buffering agents, soothing agents, preservatives, and coloring agents. It can also be used as a compounding agent.

Animals to which the antitumor agent of the present invention is administered are not particularly limited,
Targets include not only humans but also various mammalian animals such as mice, rats, dogs, cows, horses, goats, sheep, rabbits, and feral animals.

Administration to these animals and humans can be carried out by conventional routes of administration, such as intramuscular, subcutaneous, intracranial, or intraperitoneal injection.

The dose and frequency of administration are appropriately selected depending on the animal species, route of administration, purpose of administration (therapeutic purpose), etc., so that the dose is approximately 1/l to 5 mff/Kg each time, or the amount of administration is determined depending on the animal species, route of administration, purpose of administration (therapeutic purpose), etc. Another major benefit of the present invention is that certain protein substances have extremely low toxicity, so there is no need to strictly control the dosage.

[Examples] The protein substances of the present invention will be explained in more detail in Examples, but the present invention is of course not limited to the description of Examples below.

Example T p The intraperitoneal cavity of a mouse on the second day of infection with the RH strain was washed with Hank's slow I solution (+-IBSS), and the cloves contained therein were centrifuged (750G, 10 minutes, 4°C) To separate. Since the sediment contains impurities such as peritoneal cells, it is filtered using a nylon mesh ram to remove as much as possible. The obtained Tr) insect body suspension was centrifugally washed three times with HBSS solution (750G).

for 10 minutes at 4°C). 2× clover insect bodies per 1 d of sediment
Mix pre-sterilized water so that there are about 10 pieces, stir, and freeze and thaw three times using a -70°C freezer.

Then, ultrasonication (40W).

1 minute, Wakenyaku Ce1l Disru
pter Model W-220F) five times to crush as many insect bodies as possible. Centrifugation from extracted lysed antigen (16,
000G, 60 min, 4°C) to remove insoluble matter, and make the resulting upper solution isotonic by adding an equal volume of 1.7% sodium chloride solution.

The isotonic solution thus adjusted is centrifuged at ultrahigh speed.

(144,000 ets, 120 minutes, 41°C), collect the upper end, filter sterilize it, and dry it in vacuum and air to obtain the target protein substance (TLA), which is stored in a cool dark place and covered.

Physicochemical properties of TIA prepared and isolated in this way,
The characteristics of the Haku are as follows.

(1) Color and properties Pale yellow powder.

(2) Water-soluble Easily soluble.

(3)　I)H A 1% by weight aqueous solution shows r)H6,4-6.5.

(4) Molecular weight and purity Gel filtration method [GPC, column Gel-1) a, ck
Gl-W550.10.7mmφx 300mm.

Eluent r) H7, 20mM phosphate buffer (500mM
Contains NaCl), flowing! The molecular weight of the main component of TIA was confirmed to be in the range of 15.000 to 20.000 from the elution pattern of known substances using a Hitachi High Performance Liquid Chromatograph Model 655 UV 280 nm].

In order to examine the degree of partial purification, the same sample was subjected to ion exchange chromatography [column Qel pack.

GL-55D, 8mmφX100m, diethylamino group-containing, eluent A: l) l-18, 20mM1-
Lys-hydrochloric acid buffer, eluent B: solution + 500mM NaC
Adopts linear gradient 1 ~ method that completes A100% → 13100% in 30 minutes with +, flow rate 1, 0 mf
f/min, Hitachi High Performance Liquid Chromatography Instrument Fl 655
When measured with UV 200-300nm, it was 95
A single peak was observed with a purity of % or higher.

(5) Color reaction Regarding 0.1% by weight aqueous solution, phenol sulfuric acid reaction, [
1-phosphorin method and ninhydrin reaction test l~
When carried out, it was confirmed that both were positive and 1) 1, and that they were protein substances containing sugars, peptide bonds, and amino acids.

(6) Protein content The protein content in TIA was determined from a calibration curve prepared using casein according to the Folin-Lowry method and was approximately 90%.

(7) When a 0.1 W/V% aqueous solution of Ultraviolet Absorption Spectrum 1 to Trial 1 was prepared and measured with a Hitachi Ultraviolet Absorption Spectrum 1 to Lophotometer Model 200-20, the result was 26 Onm as shown in Figure 5. A spectrum with maximum absorption near 278 nm and a shoulder at 278 nm was obtained.

(8) Nucleic acid content determined by liquid chromatograph (Hitachi 655-A model).
It was found that A contained mainly hyboxanthin, UMP, ΔMP, and GMP in small amounts (1% or less).

(9) Stability T
Dissolve LA (4℃...A, room temperature...B), p1
= 1 and no abnormality was observed in the color reaction test.

In addition, A and B each have 10'Oμ9 of l-MO(
0.1 mε) as an emulsion for each of three normal mice.
1 x 102 doses per day
/head inoculation and compared with the control, three control normal mice died in 11 to 12 days, one out of three mice in groups A and B each died and two survived.

In other words, it is important to prevent infection by
16 = Activation effect did not disappear due to immune modulator effect.

Furthermore, even when a 1% by weight aqueous solution was heated at 37°C for 1 hour, the immunostimulatory effect did not disappear.

[Brief explanation of drawings]

Figure 1 (a), (b) and (C) are ICR/JCI
FIG. 2 is a graph showing the antitumor effect of the protein substance of the present invention on homogeneous tumors (S-180) in 8- to 10-week-old male mice and the effect of the combination with obioactin. ) is ICR/JCI series 4~
FIG. 3 is a graph showing the antitumor effect of the protein substance of the present invention on <8180 in 5-week-old male mice;
/c is a graph showing the inhibitory effect of the protein substance of the present invention on the engraftment of syngeneic tumors (MC-induced tumors) in male mice.
ar is a graph showing the effect of the protein substance of the present invention on the induction and suppression of autologous tumors in Table 1F rats;
The figure is an ultraviolet absorption spectrum diagram of the protein substance of the present invention. State house dome B o o o. Denpa E ↓ Go ≦ Interstate 1iI#? 〒 gBB< Figure 4 (a) Figure 5 0 and yield (%) 40'°□ O.

Claims (1)

[Scope of Claims] 1) A protein substance with a main component molecular size of 15,000 to 20,000, obtained as the upper end by centrifuging a water-soluble component obtained by physically crushing Toxoplasma gondii at 100,000 G or more. 2) An antitumor agent whose active ingredient is a protein substance with a main component molecular weight of 15,000 to 20,000, which is obtained as the upper end by centrifuging a water-soluble component obtained by physically crushing Toxoplasma gondii at 100,000 G or higher. 3) The antitumor agent according to claim 2, which is used in combination with obioactin.