JPS6287525A - Antitumor agent containing novel lymphotoxin - Google Patents

Abstract

PURPOSE:An antitumor agent, containing a novel lymphotoxin and human interferon-gamma and having more remarkably improved antitumor activity than a composite pharmaceutical containing lymphotoxin derived from lymphoblastic cells and interferon-gamma. CONSTITUTION:An antitumor agent containing a novel human lymphotoxin expressed by the formula (X is Met, H, etc.; Y is saccharide chain or H) and human interferon-gamma, preferably at 20:1-1:20 compounding ratio. The lymphotoxin expressed by the formula is a new type different from an amino acid sequence of lymphotoxin derived from lymphoblastic cells, e.g. reported by Gray, etc. The antitumor activity is remarkably higher than a conventional agent and the agent is capable of exhibiting synergistic effect with the interferon-gamma. The titled antitumor agent is applicable to malignant tumors, e.g. mammary cancer, lung cancer, gastric cancer, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Applicability) The present invention relates to an antitumor agent containing interferon-γ and a novel lymphotoxin having an amino acid sequence that is clearly distinguishable from others. More specifically, novel lymphotoxin with antitumor activity and interferon-γ with the same effect.
The present invention relates to an antitumor agent containing as an active ingredient, and the coexistence of both lymphokines has a synergistic effect on antitumor activity.

(Prior art) Lymphotoxins are induced and produced inside and outside cells by acting on sensitized lymph cells with antigens or mitogens such as phytohemaagglutinin and concanavalin A, or are produced in certain ways. It is a proteinaceous substance produced from a cell line derived from lymphocytes, which increases during culture, and is a type of lymphokine defined as a substance exhibiting cytotoxicity.

It has been reported that lymphotoxin of biological species is not a homogeneous protein in vitro, but subtypes that differ in molecular weight, charge, and stability exist.

For example, in humans, more than five types of lymphotoxin have been identified, the representative one being αLT (molecule f117
~90,000), βLT (molecular weight 30,000 to 50,000), 9γ-LT (molecular weight 12,000 to 20,000), a-heavy LT (molecular weight 140,000 to 160,000), LT complex (molecular weight 200,000 or more, etc.) (J, J, J) Evlin et al. Lymphokine, Vol. 7, p. 313 (1984)
) Academic Press Publishing]. Lymphotoxins are involved in the regulation of the immune system [Evans, C.I.
Cancer Immunology and Immunotherapy: x, (Cancer imm, and i
mmunothera-peutics) Volume 12 1
81 (1982)], or in vivo, in
Both in vitro [Papermas-ter et al. Research Communication in Chemical and
) Kusology and Pharmacology (Res, C
omm, in Ohem, path, andI
'harmacology) Volume 8, Page 413 (197
4 years).

I'apermaster clinical irnmu 70
no, andimmunopathology)
Volume 5, page 31 (1976), I'aperma
ster, Annual New York Academy of Sciences (Ann, N.W.

Acad, Sci, ) Volume 382, Page 451 (1
979).

Khan Ra, Hematology and Oncology (
■Iematology and oncology
), 11 volumes.

p. 128A (1981), Gately et al. Immunology, Vol. 27, p. 32 (19
1976).

Rosenberg et al., Journal on Immunology (J, Immunol), Vol. 110, p. 1613 (1973), Sawada et al., Japanese
Journal on Mechiisu 7 (Japanese J
, of Med, ) vol. 46, p. 263 (1976)
], it acts in a cytolytic manner or suppresses the proliferation of tumor cells. Furthermore, this effect under in vitro conditions has been reported to be more potent in tumor cells than in normal cells obtained from the same species [William et al., Cell Immunology, Vol. 6, 17
1 page (1978), Evans et al. Cancer Research (Cancer B, es,) Vol. 35, 103
5, (1975)], and because of these functions, it has been a substance that has been expected to be a therapeutic agent for malignant tumors since its inception.

Interferon is an antiviral active substance discovered by Isaacs and Lindemann in 1975, and is divided into three types: α, β, and γ, due to differences in the cells in which it is produced, its induction method, and its physicochemical and immunological properties. Species are known [Science of interferon by Shigeyasu Kobayashi, 6+l Dansha Scientific Publishing]. Further, as a result of subsequent research, various types of interferon were found to have antitumor activity in addition to antiviral activity, and in particular, γ-type interferon (
(hereinafter referred to as interferon-γ) is the α type.

It is expected to be used as an antitumor agent because it has stronger antitumor activity than the β type.

Previous research has shown that lymphotoxin, interferon-
Although both γ and γ are expected to be used as antitumor agents, the only way to produce them is to induce them from human lymphocytes or human B''!-visceral cells, and it is difficult to obtain human cells. Its exact biochemical properties remained unclear because it was difficult to purify due to the small amount of lymphokine 1 that it induces, and because it is physicochemically unstable.

However, recently, a method for easily producing Oyu using recombinant DNA technology has been developed, and it is being confirmed that it is useful as a therapeutic agent for malignant tumors.

The present inventors also used recombinant DNA technology to develop a method for producing natural interferon-γ with sugar chains in animal cells (Japanese Patent Application No. 119648/1989), and Gray et al. The reported lymphoblastoid cells (RPM
Amino acid sequence of lymphotoxin derived from I strain 1788) CGray et al., Nature 31
2, p. 721 (1984)], a new type of lymphotoxin (hereinafter referred to as lymphotoxin) with an amino acid sequence clearly distinguishable from the animal 1 ((■) cell and microbial cells was discovered, and As a result, this new lymphotoxin has significantly higher in vivo and in vitro antitumor activity than other previously reported lymphotoxin. It is confirmed that it is high.

By the way, in 1983, Williams et al. revealed that interferon-α exhibits a synergistic effect with lymphotoxin in an in vitro cell growth inhibition experiment, suggesting a synergistic effect between other β- and γ-type interferons and lymphotoxin [Williams et al. and others,
Journal Onimmunology, Volume 130, Page 518 (
1983)). This discovery has drawn attention to the possibility that combination preparations of interferons and lymphokines may act more strongly as antitumor agents than either agent alone, and a synergistic effect with interferon-γ has also been confirmed. : Williamson et al., Proceedings of the National Academy of Sciences (Pr.
o, N, A, S,) Volume 80, page 5897, (
1988), Japanese Patent Publication No. Sho 5o-s94a4].

(Means to solve the problem) During the development of a new lymphotoxin antitumor agent, the present researchers decided to use a combination agent with natural interferon-γ, which was previously reported to be derived from lymphoblastoid cells. Ajalul et al.'s combination of lymphotoxin and interferon-γ
n vitro cell growth inhibition test results (Unexamined Japanese Patent Publication No. 1987-1999-
89434), and confirmed that this composite agent has excellent clinical application as a therapeutic agent for malignant tumors, thus completing the present invention.

The content of the present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples. For example, as a specific example of the lymphotoxin of the present invention, sugar chain-bound lymphotoxin-K produced in BH cells may be used, or sugar chain-free lymphotoxin-K produced in Escherichia coli and these two white matter may be used. There is no problem even if the amino acid at the N-terminus of the amino acid is an analog type. Similarly, interferon-γ can also be used even if it is a substance that does not have a sugar chain attached to it. In addition, in the present invention, as an example of tumor cell growth inhibition, Hira (T (eLa) cells derived from cervical cancer are used, but the disease for which the new lymphotoxin and 1-interferon-r combination preparation is in competition is due to this combination. It can be applied to any tumor that is sensitive to the preparation, for example f
L cancer carcinoma lung cancer liver cancer, bladder cancer, stomach cancer, kidney cancer, leukemia, lymphoma.

Examples include malignant tumors such as brain tumors.

The composition ratio of lymphotoxin and interferon-γ in the composite preparation of the present invention is 100,000/1 to 1/
100,000 is possible, but preferably 20/1~
It has been determined that it is within the range of 1/20, but this is just a prediction from the examples (!1), and the present invention is not limited to these numerical ranges. It will be appreciated that the combination preparation may be clinically administered in the form of an intratubular or intraintrainjectable injection with a pharmaceutically acceptable carrier, or may be administered directly to cancerous tissue. Although considered appropriate, oral and other administration methods may also be used, including but not limited to, and the dosage may further vary depending on the administration route, tumor type, and symptoms.

It will be determined by your doctor as appropriate depending on your body weight, drug sensitivity, etc. Therefore, the net shape of the composite preparation can be any appropriate shape depending on the administration method, and solid,
Semi-solid or liquid forms, such as injection tablets, pills, capsules, powders.

A net form such as a solution or a suspension can be used.

(Example 1) Inhibition of cell growth by lymphotoxin-K and interferon-γ alone 12-well multiplate (1×
Hila (I (eLa) cells (I(eLa)) seeded at a cell number of 10
24 hours after planting,
50-400 U per m1 of lymphotoxin-K (I3H
1 ml of Eagle M E 3-ji (made in Japan) containing 5% tallow baby serum containing 1 to 200 U/ml of interferon-γ (CIIO-KlS, 1) produced in 1 cell) and cultured for 3 days. After culturing, 0.
Cells were collected by treatment with a 0.02% EDTA-0.25% trypsin solution, and after staining with trypan blue, the number of viable cells was measured. Figure 1 shows the ratio of the number of viable cells in each lymphokine-containing plot expressed as a percentage when the number of viable cells in the control plot that does not contain lymphotoxin-K or interferon-γ is taken as 100%. - about 50 U/m
No cell growth inhibition effect was observed up to a concentration of 50
The concentration showing % cell growth inhibition was approximately 300 lJ/ml. Furthermore, no effect was observed with interferon-γ up to a concentration of 4 U/ml, and the concentration showing 50% cell growth inhibition was 20 U/ml.

(Example 2) Synergistic effect between lymphotoxin and interferon-γ The most standard example of synergistic effect between two or more drugs is
In addition, one of the accepted methods is to use a combination that is one-fourth or less of the MIC (minimum inhibitory concentration) of each drug.
is that the desired result is obtained (Goodman
, A, et al., The Pharmacological Basis
On Therapeutex (The Pharmaco)
logical basis of therapy
ics), ? Published by Kusimilian Publix].

Therefore, the cell growth inhibition effect of simultaneous addition of a concentration of 10 to 20 U/ml, which is about one-fourth of the amount observed as MIC for lymphotoxin, and a concentration of 1 to 4 U/ml of interferon-γ was investigated in this example. It was investigated using the same method as 1. As a result, as shown in Figure 2, both lymphokines have M
Lymphotoxin-K at a concentration less than one-fourth that of IC
Even in the treated group with LOU/ml and interferon-γIU/ml, nearly 25% inhibition of cell proliferation was observed.
An extremely strong synergistic effect of lymphotoxin and interferon γ on blastocyst proliferation was found.

[Brief explanation of drawings]

FIG. 1 is a graph showing the effect of single use of lymphotoxin-K and interferon-γ on cell proliferation. FIG. 2 is a graph showing the synergistic effect of lymphotoxin and interferon-γ on cell proliferation. Patent applicant Kanebuchi Chemical Industry Co., Ltd. 1st LT, (ulrr++) Figure IFN-1 (u7ml) E Coff O - ('J TODO procedure amendment (method) % formula % 1. Indication of case 1985 Patent Application No. 230570 2, Title of the invention: Novel lymphotoxin-containing anti-ulcer drug 3, Relationship with the person making the amendment: Patent issuer address: 3-2-4 Nakanoshima, Kita-ku, Osaka, Agent

Claims (4)

[Claims] (1) Contains human lymphotoxin and human interferon-γ represented by the general formula [There is an amino acid sequence] (where X represents [there is an amino acid sequence] Met or H. Y represents a sugar chain or H) An anti-tumor agent. (2) The antitumor agent according to claim 1, wherein the human interferon-γ is a sugar chain-bound type. (3) Human lymphotoxin and human interferon-γ
2. The antitumor agent according to claim 1, wherein the unit ratio of is from 100,000:1 to 1:100,000. (4) Human lymphotoxin and human interferon-γ
The antitumor agent according to any one of claims 1 to 3, characterized in that the antitumor agent acts more synergistically on cells than either of them alone.