JPS59222422A - Antitumor agent - Google Patents

Abstract

PURPOSE:An antitumor agent comprising protein having activity like lectin as a main component. CONSTITUTION:An antitumor agent comprising a main component of protein having 188,000mol.wt. by gel filtration method, properties wherein it is divided into a subunit of 4mol having 32,000mol.wt. by SDS-gel electrophoresis and a subunit of two molecules having 30,000mol.wt., characteristics wherein it occurs in the humor of a representative of a larva of Sarcophaga peregrina when it is injured, occurring in the humor of its chrysalis at the early stage of pupation, having activity like lectin. The protein is observed to induce production of interferon of human leukocyte. Its dosage form is properly injection for intramuscular injection or intravenous injection, etc. or suppository to be used with an absorption promotor, etc. A dose is properly 3,000-5,000mug/kg/day.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antitumor agent containing protein as a main component. More specifically, the present invention naturally includes, for example, Sarcophaga pera5rina.
It is produced in the body fluid of a larva when its body surface is injured, or it appears in the body fluid of a pupa in the early stage of octopus development.
This invention relates to an antitumor agent whose main component is a protein with lectin-like activity.

In general, insects, and other invertebrates as well as higher vertebrates, injury effects include secondary changes including protein synthesis during tissue injury, such as the appearance of antibacterial active substances and cell aggregating proteins in body fluids. is known to occur. This is thought to be the result of some kind of biological defense mechanism being activated in response to external stimuli such as injury, but it plays a particularly important role in the biological defense of invertebrates that do not have the ability to produce antibodies. It is thought that there are. It has been revealed that when the body surface of the larval fly larva is injured, both a protein with significant antibacterial activity against Escherichia coli and Bacillus subtilis and a protein with lectin-like activity appear in the body fluid. (See, for example, Mizuno et al., eds., Mechanisms of Biological Defense, pp. 41-54, published by the University of Tokyo Press, October 1980). Regarding the latter protein with lectin-like activity,
Its molecular weight, spunit structure, etc. have been clarified, and in vitro, it has been shown to co-infect R tumor cells (MM4 derived from C3H/He mouse breast cancer) with macrophages.
6 cells) (e.g. Nakataka et al., Gann, Mol. 73.627-632°A
August, 19 g 2). However, there is also the fact that this protein with lectin-like activity cannot destroy tumor cells alone;
It was not clear whether vo also had antitumor activity.

The present inventors discovered that the protein having lectin-like activity is
The present invention was achieved based on the discovery that it also has remarkable antitumor activity in vivo.

That is, the present invention has a molecular weight of about 188.0 as determined by gel filtration method.
00, the molecular weight is approximately 32.000 by 5DS-Glue electrophoresis.
4 subunit molecules and molecular weight of approximately go, o o o
Sarcophaga has the property of splitting into two subunit molecules,
It has the characteristic that it appears in the body fluid of a larva when its body surface is injured, and also that it appears in the body fluid of a pupa in the early stage of octopus development, and its main component is a protein that has lectin-like activity. It is an antitumor agent.

The protein of the present invention is naturally produced in the body fluid of a larval fly, for example, by injuring the body surface of the larva by a method such as penetrating the larva with an injection needle. The fluorescent white of the present invention is extracted, manufactured by M, and isolated from this body fluid, for example, by the following method.

For example, when a body fluid is applied to a 777p-s4B column, most of the proteins contained in the body fluid are recovered in the unadsorbed fraction, but after thoroughly washing the column, a 0.2 M galactose solution is The protein of the present invention is eluted when the adsorbed protein is eluted. If galactose is removed from this by dialysis or the like, a pure aqueous solution of the protein of the present invention can be obtained.

The molecular weight of the protein of the present invention was calculated to be about 18,000 by gel filtration using Blo-Gel P 300. This protein was determined to have a molecular weight of 32.0 by 8D S-glue electrophoresis. OOO and 30. It is divided into two subunits of OOO, and the density ratio of the stained image is always V:
xvc, it can be seen that it is composed of 4 molecules of a subunit of 2,000 and 2 molecules of subunit of 30,000.

The protein of the present invention has lectin-like activity, such as hemagglutinating activity. Lectin is a sugar-binding protein (-1" or glycoprotein) other than the product of an immune reaction that aggregates alveoli,
Or something that has the property of precipitating complex carbohydrates,
The protein of the present invention has such lectin-like activity.

Furthermore, the protein of the present invention is characterized in that it also appears in the body fluid of the pupa at the early stage of dancing.

The protein of the present invention is naturally extracted, purified, and isolated from the body fluid of a centipede fly that has been injured on its body surface, or from the body fluid of a pupa in the early stages of pupa. 1 There is no limitation in any way. It may be extracted, purified, and isolated from other animals, or it may be chemically synthesized.

Alternatively, it may be produced microbiologically using genetic engineering techniques.

In an in vitro test, the protein of the present invention
In addition to destroying MM46i4 tumor cells derived from C3H/He mouse mammary carcinoma in collaboration with McGinn/Gi,
In an iv test, it was confirmed that it suppressed the proliferation of ascites cancer 8180 cells transplanted into ICR mice, prolonging and curing the mice's lives.

Furthermore, it has been confirmed that the protein of the present invention induces the production of interferon in human leukocytes, which strongly suggests that the protein of the present invention can be used as an antitumor agent.

Appropriate dosage forms for the antitumor agent include injections such as intramuscular or intravenous injections, or waste preparations combined with absorption enhancers. As a formulation method, a known method can be employed using appropriate solubilizing agents, stabilizers, excipients, etc. The dosage is
Although it varies depending on the type and degree of tumor, a, ooo-5,
000μ11/kp/day is appropriate.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 (1) Collection of body fluid from nematode fly larvae The nematode fly larva can be collected by wetting its body surface with water.
Those that inhibited shell formation and synchronized with the third instar were used. To injure the body surface, the third instar larvae are placed on ice to slow their movement, placed on a glass plate, and a syringe needle is passed through the lower part of the first body cylinder. there was.

Two days after the injury, the heads of the larvae were cut off with surgical scissors and placed on ice.
The larva was pressed against the inner wall of cgL) and the body fluid was squeezed out. With this operation, approximately 20,000 larvae to approximately tsom
J's body fluids were obtained. Collect this in a Spitz tube and collect 3.00
Orpm (5 minutes, 4°C.

The body fluid cells were removed by centrifugation using a KOKSAN MongL103R), and the supernatant was stored at -80°C as a body fluid standard. When using this standard purified material, after melting the stored material in a water bath at 37°C, centrifugation using xo, ooolI (xo minutes, 4°C) The supernatant was taken.

(2) Collection of the protein of the present invention When the body fluid obtained as described above was passed through a Sepharose 4B column, most of the protein was recovered as an unadsorbed fraction (this is the result of the present invention). (Different from protein, it is a protein that has antibacterial properties).After thoroughly washing the column with physiological saline, a 0.2M galactose solution was poured to remove the adsorbed matter.
That is, the protein of the present invention was eluted. From this, galactose was removed by dialysis to purify the protein of the present invention.

(3) Properties of the protein of the present invention When both the protein-containing fractions obtained as described above were analyzed by native gel electrophoresis, a single protein band was observed, and a portion corresponding to this band was observed. When protein was extracted from the protein and its activity was measured, hemagglutination activity was detected.

In addition, this protein was separated into two subunits with molecular weights of 32,000 and 30,000 by 5DS-gel electrophoresis, and the concentration ratio of the stained image was 2:1. Ta.

Furthermore, the molecular weight of this protein was found to be approximately 18F+,000 by the glue filtration method using Blo-Ge1 p-300.

In addition, lectin-like activity (hemagglutination activity), affinity chromatography with Sephapse 4BK, native gel electrophoresis, 5DS-gel electrophoresis, and Blo-Ge
The gel filtration method using 1 P-300 is described in The Jou
rnal of Biological Chemisj
ry, Vol, 255, Nn7.

P2919-2924 (1980).

(4) Antitumor activity experiment of the protein of the present invention 1゜ICR mice were implanted with 1X101 ascites cancer 8180 cells (intraperitoneal injection), and after 1 day, the protein of the present invention in a physiological saline solution (concentration 250 μI/-) , 400μ on the belly
! Injected. Then, we calculated the short-term survival rate compared to the control group. The results were as shown in Table 1.

Table 1 Contains 400μ of solution! injection did.

Group 2 = 1007111 injections per dose.

Table 1 shows that the protein of the present invention significantly prolongs the survival of ICR mice transplanted with ascites cancer 8180 cells.

Experiment 2 Sarcoma 18G cancer cells I x 1 to 5 ICR mice
After intraperitoneal implantation of 0M, Dayl.

On Day 2, 100 pg Lp of the protein of the present invention was administered twice. And the life extension effect on the Huntroll group, and 60
Surviving mice were observed for days.

The results are shown in Table 2.

1) Saline Dayl, 2. APX22) Protein of the present invention 10 Q pg Dayl, 2 zpX2
From Table 2, it is clear that the protein of the present invention exhibits a stronger therapeutic effect than the two administrations.

(5) Interferon-inducing activity by the protein of the present invention. The leukocytes collected from the peripheral blood of a person were placed in Eagle's MEM medium containing 10% CS to a concentration of 6 x 101 cells/d, and the protein of the present invention was added thereto. and cultured at 37°C for 4 days. Seven post-culture supernatants were collected, and the titer of human interferon was measured by the cytopathic inhibition and dye uptake method, and the relative titer to the final titer of standard interferon measured at the same time was calculated.

The relationship between the relative titer of human interferon and the amount of the protein of the present invention added is shown in FIG.

FIG. 1 shows that the protein of the present invention significantly induces the production of human interferon.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the effect of the protein of the present invention on human leukocytes to induce interfep production.

Claims (1)

[Claims] 1. The molecular weight according to the glue filtration method is about 188,000,
5DS-gel electrophoresis revealed 4 subunit molecules with a molecular weight of approximately 32,000 and 2 subunit molecules with a molecular weight of approximately 30,000.
It has the property of splitting into molecules, and centinic bar-r-(Sa
rcophaga pere9rina) has the characteristic that it appears in the body fluid of the larva when the body surface is injured, and also that it appears in the body fluid of the pupa in the early stages of pupa, and it also contains a protein that has lectin-like activity. Antitumor agent as the main ingredient.