JPH03127740A - Remedy for malignant tumor - Google Patents

Abstract

PURPOSE:To obtain a remedy for malignant tumor, having enhanced antitumor action on tumor necrosis factor(TNF), containing TNF and surfactant. CONSTITUTION:A remedy for malignant tumor containing TNF including natural type, recombinant TNF and also TNF containing modified protein showing physiologically similar physiological activity as TNF and a surfactant such as Tween 20, sodium dodecyl sulfate or gum arabic as active ingredients. The preferable administration route is intravenous injection (including intravenous drip injection) of solution or suspension or administration in tumor and a dose of TNF is 1X10<4>-5X10<5>U/m<2> surface area of body/hr. Fluidity of cell membrane of tumor cell is increased by the surfactant, function of TNF is enhanced, further permeability of tumor cell is increased, nonspecific intake of TNF in cell is raised and antitumor effect is enhanced.

Description

DETAILED DESCRIPTION OF THE INVENTION A.Objective of the Invention [Field of Industrial Application] The present invention is directed to the use of tumor necrosis factor
osisF actor (hereinafter abbreviated as TNF)] (A
ingredients), and Tween 20. An antitumor pharmaceutical composition containing as an active ingredient a TNF antitumor action enhancing ingredient such as sodium dodecyl bisulfate and gum arabic [(A
The proteins listed under Components also include modified proteins that physiologically exhibit similar physiological activities.

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

Natl,Acad,Sci,,U,S,A,,7
2.3666-3670 (1975)], for example, injecting BCG into CD13WiSS mice.
Approximately two weeks after inoculation, endotoxin was intravenously injected into the mouse serum to induce meth
A sarcoma is the name given to a factor that has hemorrhagic necrosis potential.

TNF production in living organisms is observed not only in mice but also in rats,
It has also been observed in domestic rabbits and humans [Katsuyuki Haranaka, TN
F-Tumor necrosis factor-1 Ijishinposha pages 41-108 (19
84) ].

Recently, the amino acid sequence of human TNF, the ``lII gene strand sequence, has been determined [D, Pennica et al., N.
atLIre.

312, 724-729 (1985), T, 5h
irai et al., Nature, 313. 803-80
6 (1985), A.M.

Wang et al., 5science, 288
, 149-154 (1985) Clinical research (rm bed phase Ⅰ trial and phase Ⅰ trial) of genetically recombinant human TNF [hereinafter abbreviated as rHu-TNF] is being vigorously promoted. Initially, it was strongly expected that TNF would selectively show cytotoxic effects only on tumor cells without showing any harmful effects on normal cells.
Since 1986, when the use of F became possible, basic and clinical research on TNF has progressed significantly, and it is now becoming clear that TNF is an important hormone with powerful and pleiotropic effects [Ishida Na Kawai et al., Bioholonic Project Symposium Macrofurd 198
7~Tumor Necrosis Factor
.

Therapeutic Research, Vol. 7, No. 2, 231-41
4 (1987)]. For example, the effect of suppressing fatty acid metabolism in adipocytes [B, Beutler & A, Ce
ramNature, 320. 584-588 (1
986), J., S.

Patton et al., proc, Natl, Acad
,Sci,,U,S.

A,, 83.8313-8317 (1986), M
, Kawakami et al., J. Biochem.
,, 101.331-338 (1987) ],
Growth-promoting effect on normal fibrillocytes [J, ■1lcek et al.
j.

- Exp, Med, 163. 632-643 (1
986)], promotion of neutrophil adhesion to vascular endothelial cells [1-1 Pohlman et al., J., Imm.
unol,, 136. 4548-4553 (19
86), J. R. Gamble et al., proc.
N atl.

Acad, Sci,, U, S, A,, 82.8
667-8671 (1985)], superoxide secretion promoting effect by neutrophils [S, J, K
1ebano H et al. 1, JI mmunol, 136
．． 4220-4225 (1986), M.

Tsujimoto et al., Biochem, [31
ophys, Res.

Commun, , 137.1094~1100 (
1986) ], vascular endothelial cell coagulation activity enhancement effect [
P, P, Nawtoth & D, M,
5tern, J, Exp, Med, 163°740-745 (19813), M, P, Be
Vilacqua et al., Proc. Natl. Acad.
Sc+, U, S, A,, 83.4533~453
7 (1986)], osteoclastic activity enhancement effect in chondrocytes [D, R, Bertolin+ et al., Nature
, 319, 516-518 (1986), J.
5aklatvala, Nature, 322°547-549 (1986), B, M,
Tho sulfate et al., J.

Tmmunl,, 138. 775-779 (19
87)], JILl production-inducing effect [P, P
, N awroth et al., mouth.

=4 Exp, Med,, 163.1363~1375
(1986)], the production-inducing effect of prostaglandins [M.

Kawakami et al., Biochem, B1
ophys, Res.

Commun,, 141. 482-487 (19
86), J.M.

[) ayer et al., J. EXD. Med.
162. 2163-2168 (1986) 3
, Endo1-xin shock mediator action during bacterial infection [K, J, Tracey et al., 5c1enc
e, 234. 470-474 (1986), B
.

Quetler et al., 5science, 229. 8
69-871 (1985) ], exothermic action [C, A,
Dinarello et al., EXIl, Med.
, 163.1433-1450 (1986) ],
Local Schwartzmann reaction-inducing effect [B, J.

A verbook et al., J., CIin, mmu
nol, 7, 333-342 (1987)
], inhibitory effect on cytochrome P450-dependent drug metabolic activity [P, Ghezzt et al.,! 3+ochem,
Biophys, Res, Commun,, 1
36°316-321 (1986)], Depolarizing effect of sarcolemma membrane potential [K9,), Tracey et al., J. Exp, Med.

164, 1368-1373 (1986)]. 7 Zara, TNF was detected in the serum of an elderly patient who died of sepsis, and 440 U/d (r
All patients with serum levels of Hu-T NF 100 p (equivalent to 1/ttdl) or higher died. 'There are also reports pointing out [A, Wang et al., 1 ancet, supra (8
529) Feb, 14°355-357 (198
7) ]. Therefore, when TNF is used as an antitumor agent, various effects other than antitumor effects may be induced in living organisms 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. B. 1ick et al., Cancer Res.
47°2986-2989 (1987) Ko.

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

For example, in order to enhance the antitumor effect of TNF, basic research has been conducted on the combination administration of mitomycin-C [hereinafter abbreviated as MMC], adriamycin, and various antitumor chemotherapeutic agents such as cyclophosphamide. Katsuhisa Nakata et al., Cancer and Chemotherapy, Vol. 13, No. 11, 316, hoping to be recognized for its effectiveness.
8-3193 (1986) "J.

Also, interferon, especially interferon-γ [
The antitumor effect enhancement effect of TNF was also observed by IFN-γ [B, D.

William Sulfate et al., Proc, Na
tl, A cad, Sci,, U.

S, A,,80. 5397~' (1983)
, L., Fransen et al., Eur, J., Ca.
ncer & Cl1n, 0ncol, 22. 4
19-(198), W.F 1ers et al., Col.
Spring mouth arbor 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.・Exercise 7 synergistically.

On the other hand, surfactants are known to act on the cell surface and increase the fluidity of cell membranes and the permeability of cell membranes. When the fluidity of the cell membrane increases, it cooperates with the cytoskeletal system to induce activation of the cell membrane, such as the assembly of membrane proteins (patching) and the acceleration of transmembrane rotation (flip-flop) of membrane proteins.

For example, when Tween 80 is applied to ascites hepatoma cells in VitrO, the cell membrane becomes thinner and uptake of the low molecular weight chemotherapeutic drug nitromine increases [cancer cell membrane,
Edited by Hiroshi Terayama, Nankodo, 1969, pp. 226-227].

It is known that TNF exerts its effects by binding to TNF receptors present on the cell surface and being taken up into cells [for example, Naoki Watanabe, Yojibe Niitsu, Biomezoica, Vol. 3, No. 4 , pp. 358-363]
.

In this way, the environment in which the receptor exists, the cell membrane, is important for specific binding between a protein and its receptor (receptor membrane protein) and for its uptake into cells after binding. be.

8. Structure of the Invention [Means for Solving the Problems] Therefore, the present inventors believe that it is important to develop a malignant tumor therapeutic agent that combines TNF and a component that enhances the antitumor action of low toxicity TNF. From this point of view, surfactants can increase the fluidity of tumor cell membranes and enhance the function of TNF receptors, and surfactants can also increase the permeability of tumor cell membranes to prevent TNF from entering cells. By increasing specific uptake, we hope to enhance the antitumor effect of TNF and reduce its relative side effects as a ripple effect.
The present invention was completed by discovering that TNF has an antitumor effect enhancing effect on many surfactants that do not have antitumor effects.

Furthermore, the present invention was completed by discovering a surfactant that has a particularly excellent effect of enhancing the antitumor action of TNF.

The present invention is an antitumor pharmaceutical composition containing at least a tumor necrosis factor (component A) and a surfactant (component B) as active ingredients.

The present invention also includes a method for producing an antitumor pharmaceutical composition by mixing tumor necrosis factor (component A) and a surfactant (Nichisei).

The surfactant is Tobean 20. At least one selected from the group consisting of sodium dodecyl sulfate and gum arabic
Preferably, it is a type of surfactant.

In the present invention, tumor necrosis factor includes natural and recombinant TNF, and also includes modified proteins that exhibit physiological activities similar to TNF.

TNF is a polypeptide consisting of 157 amino acids, and the present invention also includes variants thereof. Specific examples of modifications include deletion of about 1 to 12 amino acids at the N-terminus of TNF, and deletion of 8 to 10 amino acids from the N-terminus.
"-3er9-A sp" is Ar.

L MS-A ro substituted, C-terminal A 1a/
Kl or 1-eu"' with other amino acids such as Trp
, -Phe, etc. are also included.

The composition of the present invention contains a solvent in addition to component A and component B.

It goes without saying that additives and the like may be included.

The unique point of the present invention is that TNF (component A), which is an antitumor agent,
By combining surfactants, which cannot originally be expected to have an antitumor effect by themselves, as an antitumor effect enhancing component (component B), a prominent antitumor effect can be enhanced.

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
This is greater than when TNF is administered alone, and there is almost no therapeutic effect when only the TNF antitumor action enhancing component (component B) is administered.

As the TNF component (component A) of the present invention, rl-1uTN
When administering F (recombinant human TNF), as is known, the maximum safe tolerable dose in a single administration is:
5x105U/ff1 (the unit represents the body surface area of the tumor-bearing mixed-breed animal; the same applies hereafter, approximately 1.5TI1.
/bOdy), preferably in the range of 1.5 to 1-5X105U/TIt [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 dosage of the TNF antitumor action enhancing component (component B) of the present invention (component B), Tou Bean 20. mono-lium dodecyl sulfate or gum arabic can be administered within a range that is physiologically acceptable to the living body. .

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

Although it varies depending on the tissue image, cycle, and previous treatment history, 1
The dosage per session starts from a low dose, for example mainly (A
blood pressure drop, thrombocytopenia, GOT/GP caused by
Until the desired reduction in tumor size or tumor cell number is achieved without causing harmful side effects such as hemolytic tendency, anaphylactic shock, etc. caused mainly by a transient increase in T (component B). It is preferred to increase the dosage gradually. Dosing schedules can vary from about 1-3 times daily to about once every 2-10 times. The dosage and administration schedule should be 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. Determined from within.

TNF: 1xlO' ~ 5x105U/y+j/hr.

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

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,
Includes dosage forms such as intravenous injections, arterial injections, subcutaneous injections, intracutaneous injections, intramuscular injections, intraperitoneal injections, intraperitoneal perfusion agents, intratumoral injections, and intratumoral perfusion agents, and does not include injections. The dosage forms include anal/rectal/colon suppositories, vaginal/uterine active ingredients,
Sublingual medication.

Dosage forms include oral mucosal patches, external ointments, skin patches, nasal mucosal sprays, throat/esophageal mucosal sprays, oral tablets, oral capsules, and oral enteric-coated tablets. Such injections are prepared using a method known per se, that is, TNF (component A) combined with (component B), which constitutes a group of antitumor effect-enhancing components that cannot originally be expected to have an antitumor effect on its own, is injected. It is prepared by dissolving or suspending it in a sterile aqueous liquid used for pharmaceutical preparations.

Furthermore, the malignant tumor therapeutic agent of the present invention includes TNF (component A),
and (component B), which constitutes the TNF antitumor action enhancing component group, is prepared by filling the powder or lyophilized product into the same or separate vial or ampoule, and suspending or dissolving it in an aqueous solution prepared separately by Kawasaki. It can be used as Examples of aqueous fluids for use on the outbound side include physiological saline, glucose solution, Ringer's solution, and isotonic fluids containing other adjuvants.

EFFECTS OF THE INVENTION According to the present invention, the TNF sensitivity of tumors is increased, 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 that.

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

2. Examples The embodiments of the present invention will be explained in detail below using various examples, but the present invention is not limited to these examples.

<Example 1> Preparation of rHu-TNF The rl-1u-TNF used in the present invention and its production method are disclosed in a previously filed patent (Japanese Patent Application Laid-Open No. 622484).
No. 98: Application filed on April 21, 1985: The product obtained by the method described in the title of the invention "Novel bioactive polypeptide" was used. Namely, rHu-TNF was used.
Cultivate E. coli into which the gene expression vector has been introduced,
Promoted production of rHu-TNF protein. After collection, E. coli was crushed by ultrasonic waves at low temperature, and 5 hira were collected from the resulting suspension.
The method of i et al. [T.

3 hirai et al., Nature, 313. 803
~806 (1985)], D.E.A.E.
It was crudely purified by 5 epharose column chromatography. The 15-rl-1u-TNF content in this crudely purified product was approximately 30%.

In addition, the previously filed patent (Japanese Patent Application No. 6216223)
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 Sepharose 4 B by a known method. An rHu-TNF monoclonal antibody immobilized affinity column was prepared, and further purification was performed to increase the purity of the crudely purified rl-(u-TNF.

The ``ro-u-TNF content'' in this purified product was about 95% or more.

Detection of anti-tumor enhancing 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. [Lymphokine
Reports Vol, 2. ed, by E, P
ick, Academic Press.

N, Y, (191110) or F, C
, Kujl.

16- Jr. and P. Cuatrecasas [J.
Immunol,, 126°1279~1283
(1981)].

The in VitrO method used by the present inventors is an improved version of these methods, and is based on TNF-derived Shiichi 929 cells (American Type Culture Collection, CCL).
1, NCTC clone 929) 17) The effect of inhibiting growth is evaluated. That is, l −
929 cells supplemented with 5v/v% fetal bovine serum (Gibco, hereafter abbreviated as FBS) Eagle's Minimum Essential Medium (Nissui Pharmaceutical Co., Ltd., hereinafter abbreviated as EMEM, the composition of which can be found, for example, in "Tissue Culture" edited by Junnosuke Nakai et al. , Asakura Shoten (1967)), and aseptically dispersed 4 Dispense at 100 individual cells/well.

Micromultiwell plates are preincubated for 24 hours at 37°C in air containing 5% carbon dioxide. After pre-culture, use Dulbecco's phosphate buffer (Nissui Pharmaceutical, hereinafter PBS).
-) or P containing a TNF antitumor effect enhancer
BS l), 100 μl of TNF was serially diluted in 11 steps to give a post-administration concentration of 10 to 10'U/d using an Etzbendorf pipette 4780.
Administer i/well.

After administration, the micromultiwell plate is incubated for an additional 48 hours at 37° C. in air containing 5% charcoal bale gas. After main culture, the culture supernatant was discarded and each well was washed with PBS (
-) After washing once with 300μ per well, add 100μ of 0 and 1% crystal violet and 1% methanol aqueous solution prepared by Kawasaki! 1/well and fix and stain cells for 20 minutes. After washing away the excess Listal Violet and drying it, apply 100μ of Crystal Violet that stains the cells! / well with a 0.5% sodium dodecyl sulfate (SDS) aqueous solution, and the three-wavelength absorbance (the difference between the absorbance at 595 nm and the absorbance at 405 nm) was measured using an ELISA analyzer model ETY = -9.
Measure at 6 (Toyo side core ■). This absorbance is proportional to the number of surviving cells. The cell growth rate is calculated from the absorbance, with the absorbance of a control without TNF being 100% and the absorbance of a blank without cells being 0%, horizontal axis + TNF dose/dose/vertical axis: complete cell growth curve. . TN corresponding to 50% of the cell growth rate of the control without adding TNF.
The concentration of F was subtracted from the regression equation of this curve to obtain the 50% effective dose (hereinafter abbreviated as ED50). Hereinafter, the effect of enhancing the in VitrO antitumor action of TNF in the present invention will be evaluated based on this ED50 comparison.

<Example 2> Effect of enhancing the antitumor effect of rHu-TNF by Tween 20 The cytotoxic effect of Tween (Wako Tsumugi Pharmaceutical) on 1929 cells was enhanced by the above-mentioned
tr.

As a result of testing according to the assay method, EDso was 48.
Above 8μg/-, the highest dose that does not show cytotoxic effects is 48.8μ! It was 7/d.

To bean 20 administration concentration was constant at 50 μg/d, rHu
- Noshi 929 cells 1 when the TNF administration concentration was varied from 10 to 10' U/d (serial 2-fold dilution in 11 steps)
The change in the cytotoxic effect on rl-l ur-r N
A comparison with the case of administering F alone is shown in Table-1.

ED5 of control (rl-1u-TNF alone administration)
0 Ha86U/d (100%), while rl-
The EDso in the case of combined administration of 1u-T N F /Tobean 20 was 68U/d (79%), so 1
~Vienna 20 is rl-1u-TNF in L929wI cells
It is clear that the sensitivity to

<Example 3> The cytotoxic effect of sodium dodecyl sulfate (Wako Tsumugi Pharmaceutical Co., Ltd.) on 1929 cells was assayed in the same manner as in <Example 1>. As a result, the EDso was 19.5 μg/d, the highest dose showing no cytotoxic effect. The amount was 2.44 μl/-.

Table 1 shows the results of testing the antitumor effect enhancement effect of sodium dodecyl sulfate on L929 cells of rl-1u TNF in the same manner as in <Example 1>, with the administration concentration of sodium dodecyl sulfate being constant at 2.5 μq/-. Ta.

The ED50 of the control (rHu-TNF alone administration) was 68/
d (100%), whereas the ED50 for rHu-TNF/sodium dodecyl sulfate co-administration was 52U.
#! (60%), it is clear that sodium dodecyl sulfate sensitizes the sensitivity of L929 cells to rto1u-TNF.

<Example 4> Effect of gum arabic on enhancing the antitumor effect of u-TNF The cytotoxic effect of gum arabic (Wako Tsumugi Pharmaceutical Co., Ltd.) on L929 cells was assayed in the same manner as in <Example 1>.
50 was 12.5 Iug/m, and the highest dose without cytotoxic effects was 6.25 mg/-.

Table 1 shows the results of examining the effect of gum arabic on enhancing the antitumor effect of rHu-TNF on L929 cells in the same manner as in Example 1, with the S degree of gum arabic administered at a constant rate of 6 rng/d.

The EDso of the control (rHu-TNF alone administration) was 86 U/
-, whereas the EDso in the case of co-administration of rHu-TNF/gum arabic was 44 U/d (51%).
It is clear that the sensitivity to NF is increased.

Table 1 Table 1 shows the control (administration of TNF alone) and TNF/TN
F is a table showing a comparison with EDso, which is an index of cytotoxic effect on L929 cells, when administered in combination with an antitumor effect-enhancing component.

Claims (2)

[Claims] (1) An antitumor pharmaceutical composition containing at least a tumor necrosis factor (component A) and a surfactant (component B) as active ingredients. (2) The composition according to claim 1, wherein the surfactant is at least one surfactant selected from the group consisting of Tween 20, sodium dodecyl sulfate, and gum arabic.