JPS59152331A - Antitumor agent - Google Patents

Abstract

PURPOSE:An antitumor agent that contains Staphylococcus leukocidine as an active ingredient, thus being effective to leukemia, particularly human myelogenous leukemia, because it dissolves myelogenous leukemia cells without great adverse effects on normal multinuclear leukocytes. CONSTITUTION:The objective antitumor agent is obtained by using Staphylococcus leukocidine as an active ingredient. Staphylococcus leukocidine is obtained from a disease-causative Staphylococcus, e.g., Staphylococcus aureus V8 strain, and a toxin having an action of leukocytolysis composed of leukocidine S and leukocidine F. Its antitumor action is caused by the synergism of them and shows very high sensitivity to cells of leukemia, especially human myelogenous leukemia. The difference in sensitivity to a variety of cells depends on the activity of phospholipase A2 in leukocyte membranes developed by the connection of leukocidine S to receptor gangliosie GM1.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antitumor agent using Staphylococcus leufcidin.

Staphylococci, especially pathogenic staphylococci, produce many enzymes and toxins outside the bacterial body. Leukocidin (lcuk)
ocidin) is one of the staphylococcal exotoxins, Va.
n der Veldc (Ce1rule, 10
, 401 (1894)) discovered, in the cultured Kerr fluid of Staphylococcus aureus isolated from human pyogenic foci, a IL-like substance that prefers and damages white blood cells, separate from the hemolytic light purple. Later, Panton et al. CLancet,
1, 506 (1932)) named this toxin leukocidin, meaning that it has no hemolytic activity and specifically lyses polynuclear leukocytes in humans and rabbits.

Woodin CBiochem, J. , 75,158
(1960): After culturing 78 strains of Staphylococcus aureus isolated from human lesions, purification of leukocidin was carried out using carboxymethyl cellulose column chromatography on the P solution to extract the two components eluted according to the salt concentration of the F component. They named it the S component and clarified that leukocidin's leukocyte lytic activity appears only when both components are used together. In recent years, Kato et al.
)+ysica Acta, 633, 33 (1
980), Infection and Immun
ity, 3 4 (2), 36 2
(1981), Infecjion a
ndImmunity rυ animals 1), 38 (1982)
) purified leukocidin obtained from Staphylococcus aureus W4V8 strain using a carboxymethyl-8ephadex O-500 column, quadratography, and zone electrophoresis, crystallized the two components mentioned above, F and S, and analyzed its physical chemistry. We are elucidating the chemical properties and leukocyte lytic effect. However, to date, there have been no examples of using this leukocidin as a medicine, for example, as a therapeutic agent for diseases such as leukemia. This is because there is no difference in the leukocyte lytic effect of leukocidin between normal polynucleated leukocytes and white leukocytes;
This is because if an attempt was made to lyse all 11L disease cells, all normal polynuclear leukocytes would also be lysed, an unfavorable result. This was confirmed by an experiment conducted by Kato et al.
ity, 34(2), 362 (1981):]
I also get 1,000-111 shi. In other words, Kato et al.
Marked with , 5. Using isolated leukocidin, human normal leukocytes and human weak myeloid white blood cells are isolated:・! Check the binding rate between the two? The experimental result is that the difference is 1/j'+d = 44.

The present inventors have discovered that the action of staphylococcal leukocidin,
In particular, as a result of a separate investigation into its anti-r+:+r tumor action, we found that the difference in susceptibility to staphylococcal leukocidin between H and J[;υ depends on the number of receptors on the R+ membrane. Tf lI'! It was found that this depends on the degree of activation of phospholipase A2 in
Moreover, they discovered the unexpected fact that Staphylococcal leukocidin exhibits extremely high sensitivity (cytolysis) to leukemia cells, particularly human myeloid leukemia cells.

The susceptibility of Staphylococcal leukocidin to myeloid leukemia is particularly high in humans; for example, using mouse myeloid leukemia cells C1498 and human myeloid leukemia cells HL-60, the minimum amount of Staphylococcus leukocidin to lyse 1 x 10 leukocytes is 250 ny at 01498, HL-6
0 and 5pg, which is 50,000 times more sensitive. On the other hand, the minimum amount of staphylococcal leukocidin that can lyse 1 x 106 normal polynucleated leukocytes in humans is 5 opg, which is /i0 of the sensitivity to myeloid leukemia cells, and it can be used to lyse myeloid leukemia cells without significantly affecting normal polynucleated leukocytes. It is possible to dissolve

These facts have not been described in any conventionally known literature, and are new findings that could not have been predicted. The present invention was completed by further investigation based on these new findings. That is, the present invention provides an antitumor agent containing staphylococcal leukocidin as an active ingredient. b) Examples of tumors to which the invention can be applied include bone tiif! It is particularly effective against leukemias such as leukemia 11'l disease and monocytic leukemia, among which human myeloid leukemia.

In the present invention, Grape TI, N40 icocidin is a blue-purple color that has a white-faced agglomerative action obtained from staphylococci, especially pathogenic staphylococci. Add # etc. method CBiochi+η1c
ae+ 13iophysica Ac1a, 63
3, 33 + (1980)) obtained by G,
Rui 1. 'White with approximately 31,000 Leukocidin S in the molecule
and the leukocisinte σ〕nlI of about 32,000 min-1'-Ki! , it's okay.

The anti-;1□I<4yA effect of the present invention is due to the joint action of leukocidin S and leukocidin I-, and the most preferred mixing ratio is 1:, but other mixing ratios are also possible. Furthermore, even if Leukocidin S is first administered and then Leukocidin F is administered after an appropriate period of time, the anti-accus effect of the present invention can be obtained; Mr. i is also included in the present invention.

The staphylococcal leukocidin of the present invention is formulated and administered in a desired form, for example, in the form of an injection (two tablets), according to a conventional method, but it is most preferably administered in the form of an injection. The dosage per dose varies depending on the type of ulcer and the form of the preparation. For example, for adult humans, it is appropriately selected between lng and 10μ7, but when administered as an injection, it is 10ag to 100g.
A range of ag is preferred, and when administered as a tablet, 0.
A range of 1μ7 to 2μg is preferred.

Reference example.

Staphylococcus aureus (5taphylococcus au)
reus) strain (ATOCNa27733) for 2 hours in toxin production medium (yeast-extraCi medium).
After culturing in 411ffl K42 at 37°C for 2 hours, a clear supernatant was obtained by centrifugation. Taking this as the starting monthly yield, first concentrate by adding zinc chloride, deionize with phosphate, and then prepare 0.05 M acetic acid mmf (f, (
Carboxymethyl-5eph equilibrated at pH 5,2)
Column chromatography was performed using a linear concentration gradient method at a concentration of sodium chloride adsorbed on adex O50 from 0.2 to 1.284. Leukocidin F is W1 at a salt concentration of 0.45 M.
Leukocidin S was eluted at a salt concentration of 0.9M. This 1:i mixture of leukocidin F and S (6+ for protein) was dissolved in saline and used in the following experiment.

Experimental example Mouse myeloid leukemia therapeutic effect 057 BL/
6 mice (female, 5-6 weeks old) were injected with JF leukemia C1,498 cells (5 x 10 cells/mouse) into mouse wounds.
A system is created in which the drug is injected intravenously and dies within two weeks. C]
, Grape R + m r obtained from 4 mice in which 498 cells were dissolved in saline for 6'i per mouse from 24 hours after transplantation.
1 icocidin (1:J mixture of leukocidin F and S)
5. Olt! It was administered intravenously.

- Mouse glue IE survival rate in case of death is shown in the diagram. Figure 1fi
One administration of 1Cj Leukocidin in 5,0tty
) and when administering 1, /, <:+ times, all were administered every 1F4. Na'I5 Experiment-1? r'F 10 mice were used. Control group (A): After transplantation of 1.49B cells, only saline was administered in the same manner. As is clear from the drawings, C]-498 cells died within 13 days after transplantation, while C1-498 cells died within 13 days.
After transferring 498 cells to 41a, leubucidin (5,0,tty
) was administered 4 times every 24 hours (IH) (B)
In this case, the gold side is looking at survival for more than 180 days. Also 01
498 cells were transplanted, and leukocidin (5,0
In the group (0) in which the drug (μg) was administered 5 times every other day, 8 patients except 2 survived for 180 days or more.

Example 1 Leukocidin 50Tng, diluted 1000 times with mannitol in advance, and mannitol 105"i4E are dissolved in distilled water for injection to make 1000 m/!. This solution is filtered with a 0.22 μm membrane filter under aseptic operation in a sterile room. The mixture was filtered and sterilized using 1000 5〃□e volume 61 vials, each containing 1 rtte, and freeze-dried using a conventional method.

The lyophilized product was a white porous solid containing 50 ng of leukocidin per bottle, and was quickly and clearly dissolved in distilled water for injection and physiological saline for injection.

Example 2 Leukocidin 10m1i' was diluted with corn starch 471 in a conventional manner to obtain a jθ-triturate, and then mixed with mannitol 442.799. Add 160f of 12% hydroxyprobil cellulose solution as a binder and knead.
The mixture was granulated through a 35 mesh sieve and dried. Calcium stearate 3f in dry matter! Mix and sieve through 35 mesh. Each tablet weighed 50 mg and was molded using a flat mold with a diameter of 5 mm to produce tablets containing 1 μ7 of leukocidin per pig iron.

[Brief explanation of drawings]

[Shi J 1 (I shows the life-prolonging effect of leukocidin administered to mice injected with myeloid leukemia cells. Applicant: Chugai Seisaku Co., Ltd.) ← (N)

Claims (1)

[Claims] I. Vinegar Wt containing Staphylococcus leucosidi〉 as an active ingredient
') The antitumor agent according to item 1. 3. The anti-U tumor agent according to claim 1, wherein the tumor is human myeloid leukemia.