JPH01186820A - Therapeutic agent for malignant tumor - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for malignant tumors by using a tumor necrotic factor (TNF) and an activity-enhancing component such as t-PA or UK, thus increasing the TNF sensitivity of the tumor to reduce the tumor size or the tumor cell number and prolong the survival period of the animals bearing cancer. CONSTITUTION:The therapeutic agent contains TNF or a modified product having a similar activity and an activity-intensifying component selected from the tissue plasminogen-activating factor (t-PA), urokinase (UK), streptokinase (SK), plasmin (PN), plasminogen (PG), lipoprotein lipase (LPL) and a modified product having similar activity in such an amount of TNF: 1X10<4>-5X10<5>U; t-PA: 1-10mg; UK: 4X10<4>-6.4X10<5>U; SK: 1X10<4>-3X10<5>U; PN: 1X10<4>-5X10<5>U; PG: 1X10<4>-5X10<5>U; and LPL: 10-10<3>U. The similar effect is obtained by treating TNF with the activity intensifying factors except LPL preliminarily.

Description

DETAILED DESCRIPTION OF THE INVENTION A1.Objective of the invention [Field of industrial application] The present invention relates to tumor necrosis factor or modified tumor necrosis factor (hereinafter collectively referred to as TNF) exhibiting activity similar to that of tumor necrosis factor and tissue plus minogen activator or tissue plasminogen activator variants exhibiting similar activity (hereinafter collectively referred to as t-PA), urokinase or urokinase variants exhibiting similar activity (hereinafter referred to as UK ), streptokinase or streptokinase variants exhibiting similar activity (hereinafter collectively referred to as SK), plasmin or plasmin variants exhibiting similar activity (hereinafter collectively referred to as PN), plus Consists of minogen or plasminogen variants exhibiting similar activities (hereinafter collectively referred to as PG) and lipoprotein lipase or lipoprotein lipase variants exhibiting similar activities (hereinafter collectively referred to as LPL) The present invention relates to an antitumor pharmaceutical composition containing as an active ingredient at least one activity-enhancing ingredient selected from the group (ingredient A).

Further, the present invention uses t-PA, UK, and SK as TNF.

The present invention relates to an antitumor pharmaceutical composition containing, as an effective active ingredient, a component pretreated with at least one activity-enhancing component selected from the group consisting of PN and PG (component B).

BACKGROUND OF THE INVENTION TNF has been described in the literature [E., A., Carswell et al., proc.
, Natl.

ACad, Sci,, U, S, ^,, 72.38
66-3670 (1975)], for example, by intravenously injecting endotoxin into co-i 5WiSS mice about 2 weeks after inoculating them with BCG). A
This is the name given to a factor that has the ability to cause hemorrhagic necrosis. TNF production in living organisms has been observed not only in mice but also in rats, rabbits, and humans [Katsuyuki Haranaka, T.
NF-Tumor necrosis factor-, Ijishinposha pages 41-108 (1
984)].

Recently, the amino acid sequence and gene sequence of human TNF have been elucidated [0, Penn1Ca et al., Nature.

'aip 724-729 (1985), T, 5h
irai et al., Nature.

313 803-806 (1985), ^, H, W
anQ et al., 5ctence.

228, 149-154 (1985)], 3i gene recombinant human TNF (hereinafter sometimes abbreviated as rHu-TNF) clinical research (clinical phase engineering trial/phase II trial)
is being vigorously promoted. Initially, it was strongly expected that TNF would selectively exhibit cytotoxic effects only on tumor cells without showing any harmful effects on normal cells, but the use of rHLJ-TNF has now become possible. 198
Basic and clinical research on TNF has progressed significantly since 2006, and it is now being discovered that TNF is an important hormone with powerful and multifaceted effects [Nakao Ishida et al., Bioholonics・Project Symposium Macrophage 19B7~rumor 5ecro
sts ractor, Serabutink Research, Vol. 7, No. 2, pp. 231-414 (1987)]. For example, the effect of suppressing fatty acid metabolism in adipocytes [B, Beutler
& A. Cerami, Nature, 320.5
84-588 (1986), J.S., PattOn
et al., Pr0C, Natl, ACad.

Sci,, tl, 3. A,, 83.8313~83
17 (198&), H.

Kawakami et al., J.Biochem,! uL
331-33B (1987), Proliferation-promoting effect on normal fibroblasts [J, Vilffek et al., J, EXp,
Wed., 163 632-643 (1986)]
, promotion of adhesion of neutrophils to vascular endothelial cells [H, PO
hlman et al., J. Immunol, 136.45
48-4553 (1986), J.R.

Ga1l) Ie et al., proc, Natl, AC
ad, SCi,, U, S, ^,, 82°8667~
8671 (1985)], superoxide secretion promoting effect by neutrophils [S, J, formerly Ebanoff et al., J
.

Immunol, 136.4220-4225 (
198B), H.

Tsuj imoto et al., Birchen, Brop.
hys, ReS, COmmUn,.

137 1094-1100 (198B)], coagulation activity hexagonal effect of vascular endothelial cells [P, P, NaWrOth
& D.M., 5tern, J.

Exp, Wed,, 163.740~745 (1
986), H, P.

Bevi lacqua et al., proc. Natl.
ACad, SCi,,υ,S,°^,.

83, 4533-4537 (1986, Osteoclast activity hexagonal action in chondrocytes [D, R, BertOI 1rl
i et al., Nature, 319 516-518 (1
986), J, 5aklatvala1Nature,
322 547-549 (1986), B, H,
Th0111SOn et al., Immunol.

y 棧yo 775-779 (1987)], I L-
1 production-inducing effect [P, P, Nawroth et al., J
, EXp, Hed,, 163 1363-137
5 (1986)], the production-inducing effect of prostaglandins [Ho, Awakan+i et al., Biochem,
Biophys, Res.

Commun,, 141.482-487 (198
6), J.M., Dayer et al., J.EXI), R.
ed, 162.2163-2168 (1986, Endotoxin shock mediator action during bacterial infection [in J. TraCeV et al., 5science,
234.470-474, (1986), B, B
Eutler et al., 5science, 229°869~
871 (1985)], exothermic action [C, ^, Din
arellO et al., J. Exp. Hed., 163
．． 1433-1450 (1986)], local Schwartzmann reaction-inducing effect [8, J, Averbook et al.
J, Cl1n, Immunol, 333-342
(1987)], inhibitory effect on cytochrome P450-dependent drug metabolic activity [P, GheZZ i et al., Bir.
chen, Btophys, Res.

Commun,, 136.316-321 (198
6)], depolarizing effect of muscle cell membrane potential [in, J, 'rr
acey et al., J. Exp, Red.

164, 1368-1373 (1986) 1, various effects have been known. Furthermore, TNF was detected in the serum of patients who died from sepsis, and 4400/d (r
Some reports point out that all patients with serum levels higher than Hu-TN FlooIXI/d died [A, Waaij et al., 1ancet, ! , (8
529) Feb, 14.355-357 (1987
)], L/ Therefore, when TNF is used as an antitumor agent, it is expected that various effects other than antitumor effects may be caused as side effects.

[Prior art] In the clinical application of TNF as an anticancer drug, although local administration has shown some effects, systemic administration has not been as effective as expected [Tetsuo Taguchi, Cancer and Chemotherapy, Vol. 13, No. 11, 3491 ~3497 pages (1986), Tetsuo Taguchi,
Biotherapy, Vol. 1, No. 1, pp. 31-37 (1987),
M. Blick et al., CanCer Red, 4
29B6-2989 (1987)].

Therefore, due to the additive and synergistic effects of the combination of TNF and other anticancer agents such as lymphocytic drugs and chemotherapeutic agents,
Attempts have been made to enhance the antitumor effect and to reduce the relative side effects that are its ripple effect.

For example, in order to enhance the antitumor effect of TNF, mitomycin-C (hereinafter sometimes abbreviated as MMC),
Basic research was conducted on the combination administration of various antitumor therapy agents such as adriamycin and cyclophosphamide, and the combination effect was observed [Katsuhisa Nakata et al., Cancer and Chemotherapy, 13
Vol. 11, pp. 3168-3193 (1986)].

Also, interferon, especially interferon-γ (
TNF caused by (hereinafter sometimes abbreviated as IFN-γ)
The antitumor effect-enhancing effect was also observed [B, D, W
i l i iamson et al., proc, Natl.
, ^cad, SCi,.

U, S, ^,, 80.5397~(1983), L
, FranSen et al., Eur.

J, Cancer & Cl1n, 0nco1. ,2
2 419-(198), W.

Fiers et al., Colorado Spring Harbor
Symposia on Quantitative
Biology, Vol, Ll, 587-595 (
1986)].

However, the effects of these attempts have not been sufficient, and since the combination drugs used to enhance the antitumor effect of TNF are themselves antitumor agents, they may be toxic not only to tumors but also to living organisms.・There was a risk of synergistic effects.

Although the mechanism of action of TNF's antitumor effect has not yet been completely elucidated, it has been suggested that TNF has a strong relationship with cellular metabolism, particularly lysothemes. In other words, it was speculated that the destabilization of lysothemes may be responsible for -I of the cytotoxic effect of TNF [for example, H, R, Ruff1! :G,E,
Gifford, Lymphokine Vol.
2. ed.

by E, Pick, Academic Press
, N, Y, 235-272 (1981), F, C
, ull, jr. &p, cuatrecasas
, cancerRes,, 41.4885-489
0 (1981), Katsuyuki Haranaka, Mebio, Volume 3, No. 2, 27
~35 pages (198B), Naoki Watanabe et al., History of Medicine, 14
2, Vol. 2 @ 105-106 (1987), Masafumi Tsujimoto, Protein Nucleic Acid Enzyme, Vol. 32, No. 5, pp. 386-395 (1987)
)].

On the other hand, TN, which is a lymphoid protein derived from lymphocytes and monocytes,
Regarding MMC, a microorganism-derived antibiotic that is thought to have a completely different antitumor action mechanism from F.
It has been shown that the combined administration of UK or dextran sulfate (hereinafter sometimes abbreviated as DS) enhances the antitumor effect and prolongs survival compared to the case of administering MMC alone. [For example, Hisanobu Niitani, Journal of the Japanese Society of Cancer Therapy, Vol. 4 Special Issue, pp. 35-39 (
1989), Hisanobu Niitani, Metabolism, Vol. 9, No. 4, 288-29.
4 (1972), Hisanobu Niitani, Modern Medicine, Vol. 28, No. 5, pp. 912-919 (1973), Takeshi Taniguchi, Journal of the Japanese Society for Cancer Therapy, Vol. 7, No. 4, pp. 290-309 (1972)]. These studies have revealed that at the early stage of tumor cell damage by MMC, there is a marked increase in intralysomeal enzymes, which is thought to be caused by lysomeal disruption of intracellular granules.

Furthermore, the destabilizing effect of PN or LPL on lysothemes was also revealed. In addition, in ■paddy, DS,
Heparin (hereinafter sometimes abbreviated as HP), insulin (hereinafter sometimes abbreviated as INS) and apolipoprotein CIr (hereinafter sometimes abbreviated as A-CII) are produced by LPL, UK, SK and It is known that t-PA hexagonizes the activity of PN. Therefore, M by UK, DS, HP, LPL or PN
The mechanism of action of MC's antitumor action enhancing effect is that MMC
It has been suggested that torisosome destabilizing agents may enhance antitumor effects by cooperatively destroying the intratumor cell lymph seams [for example, Hisanobu Niitani et al., ``Treatment of thrombosis - Current status and “The Future”, edited by Matsuzou Matsuoka et al., 97-1
p. 03, Iji Shuppansha (1981), Hisanobu Niitani, “Enhancement of the effects of anticancer drugs and targeting therapy”, edited by Shigeru Tsukagoshi et al., 1
pp. 87-193, Science Forum (1987)]
.

000-shot configuration [Means for solving the problem] Therefore, based on such knowledge, the present inventor has developed a system to enhance the activity of TNF and reduce the relative side effects that are its ripple effects. is a malignant tumor therapeutic agent that contains an active ingredient that is a combination of TNF and a low-toxicity activity-enhancing ingredient, or a malignant tumor treatment that contains as an active ingredient a component that has been pretreated with TNF and a low-toxicity activity-enhancing ingredient. From the perspective of the importance of developing antitumor agents, we believe that lysochemistry, which is thought to have completely different antitumor effect expression mechanisms, and TNF, an antibiotic, and MMC, which are thought to have completely different antitumor effect expression mechanisms, are part of the antitumor effect expression mechanism. Focusing on the similarity in that they are closely related to destabilization, we have carefully investigated the activity-enhancing effects of TNF on a large group of rhinsome destabilizing agents that do not have direct toxic or antitumor effects. Upon screening, surprisingly, not all lysosome destabilizing agents were found to have this effect, and only a limited number of drugs were found to have the effect of enhancing TNF activity, thus completing the present invention.

The unique point of the present invention is that TNF, which is an antitumor agent, and tissue plasminogen activator, urokinase, streptokinase, plasmin, and plasminogen, which constitute a group of activity-enhancing components that cannot originally be expected to have an antitumor effect by themselves, are By combining at least one component selected from the group consisting of minogen, lipoprotein lipase, and their variants exhibiting similar activities (component A), or by combining TNF with tissue plasminogen activator,
By pretreatment with at least one activity-enhancing component selected from the group consisting of urokinase, streptokinase, plasmin, plasminogen, and their modified substances exhibiting similar activities (component B), significant The reason is that the antitumor effect can be enhanced.

The appropriate content of each activity-enhancing component in the crude antitumor drug of the present invention is as follows.

In the case where the t-PA is contained, the content ratio is TN
1 to 10 mg is appropriate for FIX104 to 5X10SU. When containing the UK, the content ratio is 4X104 to TNF1x104 to 5X105L+.
~6.4 xios U is suitable. When the SK is contained, the content ratio is TNF1X104 to 5X
1X104 to 3X105U is suitable for 105U. In the case where the PN is contained, the content ratio is TNF
1x104~5X10 for 1x104~5X105U
5U is appropriate. When the PG is contained, the content ratio is 1x104 to 5x105tJ of TNF.
1X104 to 5X105 (J is appropriate. When containing the LPL, the content ratio is 1x104 TNF
10-103 tJ is suitable for ~5X105U.

By administering this therapeutic agent for malignant tumors, tumor size or tumor cell number is reduced, and the survival period of cancer-bearing animals or cancer patients is extended. The therapeutic effect of this malignant tumor therapeutic agent is
If only at least one component selected from (component A) constituting the TNF activity-enhancing component group is administered, the therapeutic effect is greater than when TNF is administered alone, and there is almost no therapeutic effect.゛As the TNF component of the present invention, rl-1u-TNF
As is known, the maximum safe tolerated dose in a single dose is 5X105 U/7F! (The unit value is 2 or less, which indicates the body surface area of a tumor-bearing warm-blooded animal, and 1.57 Ff/bodV for humans), preferably 1.
A range of 5 to 5x105 U/m is used [Tetsuo Taguchi, Therapeutic Research, Vol. 7, No. 2, 328-3.
35 pages (1987), Akira Urushizaki, Therapeutic.
Research, Vol. 7, No. 2, pp. 336-342 (1987)].

The other side of the TNF activity enhancing ingredient (ingredient A) of the present invention is t-PA, UK, which has a track record of use as a pharmaceutical.

When administering SK and PN, as is known per se, t
- For the dosage of PA, 1-10 mg/rIt/
hr, preferably in the range of 5-10mM m/hr [for example, Shigeru Ueshima et al., Biomezoica, Vol. 2, No. 7, pp. 654-659 (1987)], and for the UK dosage, 4X104-6. 4 X105 U/rd
/ times, preferably 4X104 to 1.6 X105 U/
rd/dose range is used [for example, Yu Mizushima et al., Today's Therapeutic Medicines (1986 Edition) J, pp. 223-228;
Nankodo (1986)], for the dose of SK, 1X
104U-3X105Ll/Td/hr, preferably 1-1.6X105U/yF for the first time! After intravenous infusion over 30 minutes with 200 rItl of 5% glucose solution,
Intravenous infusion at a rate of 1 to 1.6 xlO5LJ/m/hr [for example, Yu Yamamura et al., Enzyme Therapy, p. 294 Nankodo (1969)];
04 U ~5X105 U/yd/hr, preferably 3.3 X104 U/rd in 5% glucose solution 25
Inject intravenously over 60 minutes at 0 m [for example, Yu Yamamura et al., Enzyme Therapy, p. 339, Nankodo (1969)]
.

Among the TNF activity-enhancing components (component A) of the present invention, PG or LPL, which has no track record of use as a pharmaceutical, can be administered in a dosage within the range that is physiologically acceptable to the living body. For example, in the case of Japanese people, the normal serum LPL value is 9.
±3 Ll/dl (400±150 U/body
(equivalent) [Toshibe Murase et al., "Plasma Lipoproteins", edited by Haraichi et al., Kodansha University Book (1983)].

The dose of this malignant tumor therapeutic agent is determined based on the site of tumor occurrence.

Although it varies depending on histology, stage, and previous treatment history, 1
The dosage per session should start from a low dose, for example, mainly TN.
Blood pressure decrease and thrombocytopenia caused by F.

Transient increase in GOT/GPT, mainly t-PA.

The desired tumor size or tumor cell size can be achieved without causing harmful side effects such as bleeding tendency caused by UK, SK, PN and PG, and deviations from the normal range of blood sugar and serum glyceride levels mainly caused by LPL. It is preferred to increase the dosage gradually until a reduction in number is achieved. Dosing schedule ranges from 1-3 times daily to 1-3 times every 2-10 days.
It can vary up to times. The dosage and administration schedule are determined within the physiologically acceptable range for the living body, preferably within the following range, taking into consideration the symptoms, nutritional status of the patient, bleeding tendency, blood sugar level, serum triglyceride level, age, etc. be done.

TNF: 1x104~5x105U/go/h".

TNF activity enhancing ingredient t-PA: 1-10mM m/hr.

UK: 4x104 (J ~6.4x105L
l/TIt/hr.

SK: 1 x 104 tJ ~ 3 x 105 L
l/Td/hr.

PN: 1 XIO' U ~5x10” U/
TIt/hr.

PG: 1 x 1041J ~ 5 x 105 U/T
d/hr.

L P L: 10-103U/Td/hr.

A preferred route of administration of the present therapeutic agent for malignant tumors is intravenous injection (including drip injection) of a solution or suspension, or intravenous administration.

Examples of the dosage form of the therapeutic agent for malignant tumors of the present invention include injections, and examples of such injections include drip injections,
Injections include dosage forms such as intravenous injections, arterial injections, subcutaneous injections, intramuscular injections, intraperitoneal injections, intraperitoneal perfusion agents, intratumoral injections, and intratumoral perfusion agents. Other dosage forms include anal/rectal/colon suppositories, vaginal/uterine suppositories,
Sublingual medicine.

Dosage forms include oral mucosal patches, external ointments, skin patches, nasal mucosal sprays, and throat/esophageal mucosal sprays. Such an injection is prepared using a method known per se, that is, t-PA, which constitutes a group of TNF activity-enhancing components that cannot be expected to have an antitumor effect by itself.

TNF or t-PA, UK in combination with at least one component selected from the group consisting of UK, SK, PN, PG and LPL (component A).

Prepared by dissolving or suspending TNF pretreated with at least one TNF activity-enhancing component selected from the group consisting of SK, PN, and PG (component B) in a sterile aqueous solution commonly used for injections. be done.

Furthermore, the malignant tumor therapeutic agent of the present invention includes TNF and TNF.
At least one component selected from (component A) constituting the activity-enhancing component group is a powder or freeze-dried product.
Alternatively, it can be filled into a separate vial or ampoule, and when used, it can be suspended or dissolved in a separately prepared aqueous solution for injection. Aqueous solutions for injection include physiological saline and glucose solutions.

Examples include isotonic formulations containing Ringer's solution and other adjuvants.

The malignant tumor therapeutic agent of the present invention can also be prepared by pretreating TNF with a TNF activity-enhancing component (component B) under appropriate conditions.

Furthermore, the malignant tumor therapeutic agent of the present invention can also be administered by pretreating TNF with an immobilized enzyme in which a TNF activity-enhancing component (component B) is immobilized on a suitable carrier such as 5epharose 4B by a known method. It can be prepared.

Pretreatment conditions include pH 5 to 10, 0 to 60°C, and 1 minute or more, preferably pH 6 to 8.25 to 37°C.

10 minutes to 5 hours, more preferably pH 7.4.

37°C for 1 to 5 hours. Such pretreatment can be stopped by cooling or by adding a protease inhibitor such as aprotinin (hereinafter sometimes abbreviated as AP) or soybean-derived trypsin inhibitor. After pretreatment, ultrafiltration,
Components that do not contribute to antitumor effects can be removed by purification operations such as DEAE 5epharose column chromatography and anti-rHLj-TNF monoclonal antibody immobilized affinity column chromatography.

[Effect] According to the present invention, the TNF sensitivity of tumors is sensitized, the tumor size or the number of tumor cells is reduced, and the survival period of cancer-bearing animals or cancer patients is prolonged, compared to when TNF is administered alone. You can expect.

The composition, treatment method, and antitumor effect enhancement method of the present invention are extremely useful for treating tumors or preventing tumor metastasis.

EXAMPLES The effects and modes of implementation of the present invention will be explained in detail below with reference to Examples, but these are not intended to limit the present invention.

The rHu-TNF used in the present invention and its manufacturing method are described in the previously filed patent (Japanese Patent Application No. 61-90087).
No.: Application: April 21, 1985: The product obtained by the method described in the title of the invention "Novel Physiologically Active Polypeptide" was used. That is, by culturing E. coli into which the rHu-TNF gene expression vector was introduced, r)
(Promoted the production of u-TNF protein. After collection, E. coli was disrupted by ultrasonication at low temperature, and the resulting suspension was
ai et al.'s method [T, 5hirai et al., Nature,
313 803-806 (1985)], D.
Crude purification was performed by EAE 5 epharose column chromatography. The rHu-TNF content in this crude product was about 30%.

Zarani, the earlier filed patent (Japanese Patent Application 16223-1982)
No. 3: Filed on July 1, 1988: The monoclonal antibody against rHu-TNF described in the title of the invention ``Monoclonal antibodies and hybridoma cells'' was immobilized on 5epharose 4B by a known method. An rHu-TNF monoclonal antibody immobilized affinity column was prepared, and rough purification was performed to increase the purity of the crudely purified r)-1u-TNF.
The TNF content was approximately 95%.

Detection of tumor site effect of TNF Bioassay methods for measuring the cytotoxic activity of TNF include a method of measuring tumor necrosis effect in vivo and a method of measuring tumor cell cytotoxic effect in vitro.

Measurement of tumor cell damage effect using the in VitrO method is described, for example, by M. R. Ruff et al. [117m1) hokir
le RepOrtS VOl, 2゜ed, by E
,Pick,ACadelliCpress,N
, Y, (1980)] or F, C, ul
l, Jr. and p, cutrecasas [J.

Immunol, 126.1279'~1283
(1981)].

The in VitrO method used by the present inventors is an improved version of these methods, in which TNF is isolated from L-929 cells (American Type Culture Collection, CCL).
1. This test evaluates the effect of inhibiting the growth of NCTC clone 929). That is, L-929 cells were treated with 5v/v% fetal bovine serum (Gibco, FB under JX).
Eagle's Minimum Essential Medium (Nissui Pharmaceutical Co., Ltd., hereinafter abbreviated as EMEM) supplemented with Eagle's Minimum Essential Medium (Nissui Pharmaceutical Co., Ltd., hereinafter abbreviated as EMEM), whose composition is described, for example, in "Tissue Culture" edited by Junnosuke Nakai et al., Asakura Shoten (1967) ), 96
Aseptically inject 4xio: 1 cell/100 μm into a micro-multiwell plate for tissue culture (Falcon) using an Etzpendorf pipette 4780 (Yatron).
Dispense 2/well.

Micromultiwell plates are preincubated for 24 hours at 37°C in air containing 5% carbon dioxide. After pre-culture, add Dulbecco's phosphate buffer (Nissui Pharmaceutical Co., Ltd., hereinafter PBS).
(abbreviated as (-)) or PBS (-) containing a TNF antitumor effect enhancer, with a concentration of 10 to 10 after administration.
TNF, serially diluted 2-fold in 11 steps to give 4 U/me, is administered aseptically using an Eppendorf pipette 4780 at 100 μIt/well. After administration,
The micro multiwell plate is incubated for 48 hours at 37°C in air containing 5% carbon dioxide. After the main culture, the culture supernatant was discarded and each well was incubated with PBS (-
) After washing once with 300 μl/well, add 100 μm/well of 0, 1% crystal violet, 1% methanol aqueous solution prepared at the time of use, and fix the cells for 20 minutes.
dye. After washing away excess listal violet and drying, extract the crystal violet staining the cells with 100 μl/well of 0.5% sodium dodecyl sulfate (SDS) aqueous solution, and calculate the difference between the absorbance at 595 nll and the absorbance at 405 nll. Three-wavelength absorbance is measured with an ELIS^ analyzer model ETY-96 (Toyo Sokki (11). This absorbance is proportional to the number of surviving cells. The absorbance of the control without TNF is 1
00%, the cell growth rate is calculated from the absorbance with the absorbance of the blank where no cells are present as 0%, and the horizontal axis: TN
F dose/vertical axis: Create a cell growth rate curve. TN corresponding to 50% of the cell growth rate of the control without TNF.
The concentration of F was calculated from the regression equation of this curve and set as the 50% effective dose (hereinafter abbreviated as ED5o). Hereinafter, the effect of enhancing the 1nVitrO antitumor effect of TNF in the present invention will be evaluated based on the comparison of this ED5°.

The cytotoxic effect of urokinase (Mochida Pharmaceutical, "Uronase 6000 International Units") on L929 cells was assayed according to the above r@u-TN F in vitro assay method, and as a result, E'D5o was 1500 U/l.
d. The highest dose without cytotoxic effects is 375 U/r.
It was nR.

The urokinase administration concentration was kept constant at 300 U/rIil, r
Hu-TNF administration concentration is 10-104 U/rIJ! (
Fig. 1 shows the changes in the cytotoxic effect on 1929 cells when the drug was diluted in 11 steps (2-fold serial dilution) in comparison to when rHu-TNF was administered alone.

In Figure 1, the horizontal axis represents rHu-TNFII degrees, and the vertical axis represents [9
29 cell growth rate, and the plot marked O represents the control (rH
u-TNF alone administration), plots marked with * indicate combined administration. It is a graph showing the correlation between rHu-TNF dosage and L929 cell growth rate.

The second to fifth times are similar graphs.

Control (rHu-TNF alone administration) ED5Q is 100U
/d, whereas the ED5o in the case of combined administration of rHu-TNF/urokinase was 30 U/W11, indicating that urokinase sensitizes the sensitivity of L929 cells to rHu-TNF. .

Example 2 rHu-TNF due to plasminogen activity
Highly effective tissue plasminogen activator (Bioscot) for anti-tumor use
As a result of assaying the cytotoxic effect on L929 cells (human origin) in the same manner as in Example 1, the ED5o was 2.5 μg/
For rr11 or more, the highest dose that does not show cytotoxic effects is 2.
．． It was 5μ(J/rail).

The tissue plasminogen activator administration concentration was 333na.
Figure 2 shows the results of assaying the antitumor effect enhancement effect of rHu-TNF on [929 cells by tissue plasminogen activator at a constant value of /wit] in the same manner as in Example 1.

The ED5o of the control (rHu-TNF alone administration) was 100 U.
/d, whereas the ED5o in the case of co-administration of rHu-TNF/tissue plasminogen activator is 20U/d.
rni, it is clear that tissue plasminogen activator sensitizes [929 cells' sensitivity to rHu-TNF.

Example 3 Enhancement effect of antitumor effect of rHu-TNF by plasmin The cytotoxic effect of plasmin (Kabi, human origin) on 1929 cells was assayed in the same manner as in Example 1.
i, E D G;! 1.25 CU/d1 The highest dose that did not show cytotoxic effects was 313 mCU/me.

Figure 3 shows the results of testing the effect of plasmin to enhance the antitumor effect of rHu-TNF on [929 cells] in the same manner as in Example 1, with the plasmin administered at a constant concentration of 150 mCU/ml.

ED5Q of control (rHU-TNF alone administration) is 500U
/d, whereas the ED5° in the case of combined administration of rHu-TNF/plasmin was 150 LJ/me, indicating that plasmin sensitizes the sensitivity of L929 cells to rHu-TNF. It is.

Example 4 Lipoprotein lipase (Sigma L-8634
As a result of assaying the cytotoxic effect of Pseuaomonas (derived from the genus Pseuaomonas) on L929 cells in the same manner as in Example 1,
ED5o was 12.5 tJ/rIJ1, and the highest dose that did not show cytotoxic effects was 3.13 LJ/rrIi.

The lipoprotein lipase administration concentration was kept constant at 2.5 U/d, and the L9 of rHu-TNF was determined by lipoprotein lipase.
The antitumor activity enhancing effect on 29 cells was assayed in the same manner as in Example 1, and the results are shown in FIG.

The ED5o of the control (rHu-TNF alone administration) was 100L.
I/d, whereas the ED5o in the case of combined administration of rHu-TNF/lipoprotein lipase was 25 U/d, indicating that lipoprotein lipase sensitizes the sensitivity of L929 cells to rHLJ-TNF. That is clear.

Example 5 Plasminogen (Kabi, human origin) L929
As a result of assaying the cytotoxic effect on cells in the same manner as in Example 1, the ED5o was 5 CU/me or more, and the highest dose that did not show any cytotoxic effect was 625 mCU/ml.

Plasminogen administered at a constant concentration of 150 mCU/r11, tissue plasminogen activator (BiO3COt, human origin) administered at a constant concentration of 83.3 nM d, rHu-TNF by plasminogen/tissue plasminogen activator. The anti-tumor effect enhancing effect on L929 cells was assayed in the same manner as in Example 1, and the results are shown in FIG.

The ED5o of the control (rHu-TNF alone administration) was 700 U.
/d, whereas the ED5o in the case of combined administration of rHu-TNF/plasminogen/tissue plasminogen activated prisoners was 150 U/r11, indicating that plasminogen/tissue plasminogen activity The conversion factor is [9
It is clear that the sensitivity of 29 cells to rHu-TNF is enhanced.

At this dose concentration, the rHu-TNF/plasminogen or rHu-TNF/tissue plasminogen activator combination significantly reduced the sensitivity of [929 cells to rHu-TNF compared to rHu-TNF alone. No sensitization was observed.

Example 6 rHu-TNF4xlO5U/d, 10(li (solvent: PBS(-)) and plasmin (Kabi, human origin) 1210 mCtJ/rrdt, 100 μm
(Solvent is PBS (-)) After stirring and mixing, the predetermined time (0
, 1, 2, 3, 4, and 5 hours), pretreated at 37°C, and after a predetermined period of time, cooled on ice and treated with AP (Teikoku Organ Pharmaceutical Co., Ltd., Antiklein Note 25) 606 U/mff1, iooμ
m (solvent: PBS(-)) was added and mixed with stirring, and the pretreatment was stopped. The cytotoxic effect of this pretreatment mixture on [929 cells] was assayed according to the procedure of Example 1, and the results are shown in Table 1.

Table 1 shows the time required for pretreatment when rHu-TNF was pretreated with plasmin, and the time required for pretreatment of rHu-TNF with plasmin pretreatment.
- It is a table showing the correlation with ED5o, which is an index of the cytotoxic effect of TNF on L929 cells.

Following the procedure of Example 1, the post-administration concentration of rHu-TNF was 1.
2 consecutive steps in 11 steps so that 0 to 104 U/rd
Post-administration concentrations of plasmin and AP when diluted two-fold were 30 μCU/m to 30 gnCU/rrdl and 15 mu/Id to 15 U/rIIi, respectively. The highest doses of plasmin and AP that do not show cytotoxic effects are 313 mCU/mO and 81 U/ll11, respectively, and when rHu-TNF is not administered, plasmin and AP are within the administration concentration range of this example. does not show any cytotoxic effect on L929 cells.

The ED5o of the control (rHu-TNF alone administration) was 86 U/
d, whereas the ED5o in the case of plasmin pretreatment rHu-TNF administration differs depending on the pretreatment time, but in all cases it is smaller than the control case r) (
It is clear that the sensitivity to u-TNF is increased. Under the pretreatment conditions of this example, the highest rHu-TNFvLii tumor action enhancing effect was observed at a pretreatment time of 3 hours.

[Brief explanation of the drawing]

Figures 1 to 5 are graphs showing the correlation between the rl-lu-TNF dosage and the L929 cell growth rate, with the horizontal axis representing the rHu-TNF concentration and the vertical axis representing the L929 cell growth rate.

Claims (8)

[Claims] (1) Tumor necrosis factor or tumor necrosis factor variants exhibiting similar activity, tissue plasminogen activator, urokinase, streptokinase, plasmin,
A group consisting of plasminogen, lipoprotein lipase, and their variants exhibiting similar activities (A
1. An antitumor pharmaceutical composition containing at least one activity-enhancing component selected from the following as an effective active ingredient. (2) Tumor necrosis factor or a tumor necrosis factor variant exhibiting similar activity to tissue plasminogen activator;
A component pretreated with at least one activity-enhancing component selected from the group consisting of urokinase, streptokinase, plasmin, plasminogen, and their modified substances exhibiting similar activities (component B) as an active active ingredient. An antitumor pharmaceutical composition containing. (3) In the case where the tissue plasminogen activator or the modified plasminogen activator is contained, the content ratio is 1×10^4 to 5× the tumor necrosis factor or the modified tumor necrosis factor. 1~10m for 10^5U
The antitumor pharmaceutical composition according to claim 1, which is g. (4) When the urokinase or the urokinase variant is contained, the content ratio is 1 x 10^4 to 5 x 10^5 U of the tumor necrosis factor or the tumor necrosis factor variant.
The anti-tumor pharmaceutical composition according to claim 1, which has an amount of 4 x 10^4 to 6.4 x 10^5 U. (5) In the case where the streptokinase or the streptokinase variant is contained, the content ratio is 1 x 10^4 to 5 of the tumor necrosis factor or the tumor necrosis factor variant.
The antitumor pharmaceutical composition according to claim 1, wherein the amount is 1 x 10^4 to 3 x 10^5 U relative to x10^5U. (6) When the plasmin or the plasmin variant is contained, the content ratio is 1 x 10^4 to 1 x 10^4 to 5 x 10^5 U of the tumor necrosis factor or the tumor necrosis factor variant. The antitumor pharmaceutical composition according to claim 1, which has an amount of 5x10^5U. (7) When containing the plasminogen or the modified plasminogen, the content ratio thereof is 1×10^4 to 5×10^ of the tumor necrosis factor or the modified tumor necrosis factor.
The antitumor pharmaceutical composition according to claim 1, wherein the amount is 1x10^4 to 5x10^5U per 5U. (8) When containing the lipoprotein lipase or the lipoprotein lipase variant, the content ratio is 1 x 10 of the tumor necrosis factor or the tumor necrosis factor variant.
The antitumor pharmaceutical composition according to claim 1, wherein the amount is 10 to 10^3 U compared to ^4 to 5 x 10^5 U.