JPH03123735A - Therapeutic agent of malignant tumor - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent of malignant tumor containing tumor necrosis factor(TNF) and a proteolytic enzyme as ingredients for enhancing antitumor action of TNF. CONSTITUTION:The objective therapeutic agent containing as the ingredients for enhancing antitumor effect a combination of TNF of both natural TNF and a recombinant TNF and a proteolytic enzyme, preferably trypsin or papain, incapable of expecting the own antitumor effect if singly used. The preferable dosing route of this therapeutic agent is by intravenous injection (including infusion) of a solution or suspension thereof or by intratumor injection and the dose as TNF is 1X10<4>-5X10<5>U/ml surface area of body/hr. The proteolytic enzyme is used at a dose within a range physiologically acceptable to living organism.

Description

DETAILED DESCRIPTION OF THE INVENTION A.Objective of the invention [Industrial field of application] The present invention relates to tumor necrosis factor [Tun+or
osisF actor (hereinafter abbreviated as TNF)] (A
An antitumor pharmaceutical composition containing at least one proteolytic enzyme (component B) selected from tribium, papain, etc. as a TNF antitumor action-enhancing component [
The proteins shown in (component A) and (component B) also include modified proteins that exhibit physiological activities similar to these.

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

Natl,Acad,Sci,,U,S,A,,7
2.3666-3670 (1975)], for example, injecting BCG into CD-13w1ss 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 and gene sequence of human TNF have been elucidated [D, Pennica et al., Nat.
re.

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)], and clinical research (clinical phase 1 and phase Ⅰ trials) of recombinant human TNF [hereinafter abbreviated as rHu-TNF] is being vigorously promoted. Initially, T.
NF does not show any harmful effects on normal cells,
Although it has been shown that TNF has a strong cytotoxic effect selectively only on tumor cells, basic and clinical research on TNF has continued since 1986, when it became possible to use Trl1l-TN and F. has made remarkable progress, and it is now becoming clear that TNF is an important hormone with powerful and multifaceted effects [Nakao Ishida et al., Bioholonic Project Symposium, Macropherd 1987-
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 & △, Ce
rami.

Nature, 320. 584-588 (1986
), J, S.

Patton et al., Proc. Natl. Acad, S.
ci,,U,S.

A,,83. 8313-8317 (19El
et al.'), M., KaWakalli et al.
, J., Biochen+,, 101.331-33
8 (1987)], proliferation-promoting effect on normal fibrotic cells [
J, V 1lcek et al., J.

Exp, Med, 163. 632-643 (1
986)], promotion of adhesion of neutrophils to vascular endothelial cells [H.

Pohlman et al., J. mmuno1. ．． 1
36. 4548-4553 (1986), J, R
, Gamble et al., Proc, Natl.

Acad, Sci,, U, S, A,, 82.8
667-8671 (1985)], promotion of superoxide secretion by neutrophils [8, J, Kleba
Noff et al., J.

Immunol, 136.4220-4225
(1986), M.

TSUjimOtO et al., B 1ochen+, 31
ophys, Res.

Con+n+un, , 137.1094~1100
(1986) ], coagulation activation hexagonism of vascular endothelial cells [P, P, Nawtoth & D, M, 5
tern, J, Exp, Med,, 163°7
40~γ45 (1986), M, P, Bev
ilacqua et al., Proc. Natl. Acad, S.
ci,,jJ,s,A,,83.4533-4537
(1986) ], osteoclastic hexagonal action in chondrocytes [D, R. Bertolini et al., Nature.
319,516~51g (198B>, J, 5
aklatvala, Nature, 322°5
47-549 (198B>, B, M,
Thomson et al., J.

l1luon1. , 138. 775-779 (198
7)], IL-1 production inducing effect [P, P
, Nawroth et al., J.

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

Kavakami et al., B 1ochea+, B 1
ophys, Res.

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

[) ayer et al., J. Exp, Mad.
162. 2163-2168 (1986)]
, endotoxin shock mediator action during bacterial infection [K, J, Tracey et al., 5science
, 234. 470-474 (198B), B.

BeLItler et al., 5science, 229.
869-871 (1985) ], exothermic action [C, A
, Dinarello et al., J., EXIT, Me.
d,, 163.1433-1450 (1986)
], local Schwartzmann reaction-inducing effect [8, J.

A verbook et al., J. [)Iin, lm1un
o1. ．． 7. 333-342 (1987) ],
Inhibition of cytochrome P450-dependent drug metabolic activity 11i
11 effects [P, GMZZi et al., Biochem,
Biophys, Res, Commun,, 1
36°316-321 (1986) ], Muscle Cell II! The depolarizing effect of potential [K, J, Tracey et al., J, Exp, Med.

164, 1368-1373 (1986)]. Furthermore, TNF was detected in the serum of patients who died from sepsis, and 440 U/d (r
Hu-TN F 100 pg/dc equivalent) or more (7
) Some reports point out that all patients with blood mL'bell died [A, Wang et al., 1 ancet, supra (8529
) Feb, 14°355-357 (1987)
]. Therefore, when using TNF 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, local administration has shown some effects, but systemic administration has not been as effective as expected [Tetsuo Taguchi, Cancer and Chemotherapy, 13
Vol. 11, pp. 3491-3497 (1986), Tetsuo Taguchi, Biotherapy Vol. 1, No. 1, pp. 31-37 (1987)
, M.B. 1ick et al., Cancer Re
s, 47°2986-2989 (1987)].

Therefore, attempts are being made to enhance the antitumor effect through the additive/synergistic effect of the combination of TNF and other anticancer agents such as cytotoxic agents and chemotherapy agents, and to reduce the relative side effects as a ripple effect. has been done.

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)].

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

W i+ +iamson et al., Proc, Na
tl,,A,cad,3 ci,,IJ.

S, A,,80. 5397~(1983),
L., Fransen et al., Eur., J., Ca.
ncer &Cl1n, Qnco1. , 22. 419
~(198), W. F 1ers et al., Col.
spr+n.

l-1arbor 3yIlposia on Qu
antitative 3 iology.

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 synergistically.

On the other hand, proteolytic enzymes are known to act on the cell surface and change the microscopic state of the cell surface.

For example, it is known that the negative charge of some cells is derived from sialic acid, and trypsin acts on cell surface proteins, liberates sialic acid, and reduces the negative charge on the cell surface. Cell Membranes, edited by Hiroshi Terayama, Nankodo, 1969, pp. 106-108].

In addition, papain also acts on cell surface proteins,
Releases certain polysaccharides and reduces positive charges on the cell surface [Cancer Cell Membrane, edited by Hiroshi Terayama, Nankodo, 1969,
143-144].

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]
.

It has been pointed out that the micro-charged environment on the cell surface is important for the non-specific encounter between cells and proteins, which is the prelude to specific binding between proteins and their receptors. Cell membrane, edited by Hiroshi Terayama, Nankodo, 1969, 141-
144 pages].

Composition of the Invention [Means for Solving the Problems] Therefore, the present inventors believe that it is important to develop a therapeutic agent for malignant tumors that combines TNF and a component that enhances the antitumor action of low toxicity TNF. From this point of view, by controlling the microscopic charge state on the surface of tumor cells using proteolytic enzymes, we can enhance the binding between TNF and receptors, enhance the antitumor effect of TNF, and reduce the relative side effects as a ripple effect. The hope is that there will be no direct toxicity.

Regarding many proteolytic enzymes that have no antitumor effect, TN
As a result of intensive screening for the effect of F to enhance anti-tumor action, it was found that TNF had an effect of enhancing anti-tumor action, and the present invention was completed. TNF is also an excellent proteolytic enzyme.
The present invention was completed based on the discovery of a substance that has the effect of enhancing antitumor action.

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

Proteolytic enzymes also include modified proteins that exhibit physiological activities similar to these. In particular, it is preferable that the protease is at least one selected from the group consisting of trypsin and papain.

Tumor necrosis factor (TNF) includes natural TNF, recombinant TNF, and also includes modified proteins that exhibit physiological activities similar to these.

The recombinant type also includes those modified to the extent that the antitumor activity of TNF is not impaired.

Modifications include deletion of amino acids, substitution with other amino acids, and the like. Specifically, N-terminal 1 to 1
The 8th to 10th pro8-3 er9-A sp” from the N-terminus was deleted by Ar(1
-L VS-A r (substituted with l, C-terminal A l
a d6 or eu with other amino acids (e.g. T
r+), Phe, etc., or a combination thereof.

It goes without saying that the composition of the present invention may contain solvents, additives, etc. in addition to component A and component B.

The unique point of the present invention is that TNF (△ component), which is an antitumor agent,
By combining proteolytic enzymes, which cannot originally be expected to have an antitumor effect on their own, as an antitumor effect-enhancing component, it is possible to achieve an enhanced antitumor effect.

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 (day component) is administered.

As the TNF component (component A) of the present invention, rHu-TNF
As is well known, the maximum safe tolerable dose in a single administration is 5xlO'U/m (unit: 0' indicates the body surface area of a tumor-bearing phlebotomy animal; similarly, in humans, 1
．． 5rd/body), preferably 1,5
A range of ~5x105LI/m is used [Tetsuo Taguchi,
Therapeutic Research, Vol. 7, No. 2, 328-33
5 pages (1987), Akira Urushizaki, Therapeutic.
Research, Vol. 7, No. 2, pp. 336-342 (1987)]
.

The dose of trypsin or papain (component B) that enhances the TNF antitumor action of the present invention can be within a range that is physiologically acceptable to the living body [Yamamura Yu-ed., Enzyme Therapy, Nankodo, 1969, 10.
pages 212-218].

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 dose starts from a fixed amount, 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 bleeding tendency, anaphylactic shock, etc. caused mainly by (component B) due to a transient increase in T. 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 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: IX10” ~5X105Ll/TIt
/hr.

The preferred route of administration of the present therapeutic agent for malignant tumors is intravenous injection (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,
Injections include D-pulse injections, arterial injections, subcutaneous injections, intracutaneous injections, intramuscular injections, intraperitoneal injections, intravenous perfusion agents, intratumoral injections, intrais perfusion agents, etc. Other dosage forms include anal/rectal/colon suppositories, vaginal/uterine suppositories, and sublingual suppositories.

Includes dosage forms such as 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 usually prepared by adding TNF (component A) in combination with (component B), which constitutes a group of antitumor effect-enhancing components that cannot originally be expected to have an antitumor effect on their own, using a method known per se. It is prepared by dissolving or suspending it in the sterile aqueous liquid to be used.

In addition, the malignant tumor therapeutic agent of the present invention includes TNF (hemocomponent),
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 a separately prepared aqueous solution for injection before use. It can be used as Aqueous solutions for injection include physiological saline, dextrose, Ringer's solution, and equipolarized solutions 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 rHu-TNF used in the present invention and its manufacturing method are described in a previously filed patent (Japanese Patent Laid-Open No. 62-24849).
No. 8: Application filed on April 21, 1985: The product obtained by the method described in the title of the invention "Novel physiologically active polypeptide" was used. Namely, rl-1u-T
E. coli into which the NF gene expression vector had been introduced was cultured to promote production of rHu-TNF protein. After collecting the bacteria, E. coli was disrupted by ultrasonication at low temperature, and the resulting suspension was crushed for 5 hours.
The method of irai et al. [T.

3hirai et al., Nature, 313. 803
~806 (1985)]
Crude purification was performed by Sepharose column chromatography. The rHu-TN F content in this crudely purified product was about 30%.

Furthermore, the patent application filed earlier (Japanese Patent Application No. 16223
3M + 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. A TNF monoclonal antibody immobilized affinity column was prepared, and further purification was performed to increase the purity of crudely purified rHu-TNF.

The rHu-TN F content in this purified product was about 95% or more.

Assay of TNF's anti-tumor effect enhancing effect 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 by in vitro method is described, for example, by M. R. Ruff et al. [Lymphoktne et al.
Relorts Vol, 2. ed, by E.
Pick, Acadea+ic Press.

N, Y, (1980) ] or F, C,
KIJll.

Jr, and P, Cutreca Sas [J,
I mmunol.

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, NCT Calone 929) f7) The effect of inhibiting growth is evaluated. That is, L
-929 cells were treated with 5v/v% fetal bovine serum (Q 1bc
o, hereafter abbreviated as FBS) supplemented with Eagle's Minimum Essential Medium (Nippon Pharmaceutical Co., Ltd., hereinafter abbreviated as EMEM; its composition is described, for example, in "Tissue Culture", edited by Junnosuke Nakai et al., Chokoku Shoten (1967)). ),
Aseptically culture 4 x 103 cells/100 in a 96-well tissue culture micro-multiwell plate (Falcon) using an Etzpendorf Bibet 478ONtl Yatron).
/ 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 (Nihon Pharmaceutical, hereinafter PBS (-
), serially diluted 2-fold in 11 steps so that the post-administration concentration was 10 to 104 U/L, was aseptically diluted to 100
Administer in μJl/well. After administration, the micro multiwell plate was incubated at 31°C in air containing 5% carbon dioxide.
Then, main culture is continued for another 48 hours. After the main culture, the culture supernatant was discarded, and each well was washed once with PBS (-) 300μ/well, and 100μ/well of 0, 1% crystal violet, 1% methanol aqueous solution prepared at the time of use was added. Fix and stain cells for 20 minutes. After washing away excess listal violet and drying, the crystal violet staining the cells was extracted with a 100 μm/well 0.5% sodium dodecyl sulfate (SO3) aqueous solution, and its absorbance at 595 nIll and 405 μm were extracted.
The three-wavelength absorbance of the difference in absorbance at m is measured using an ELISA analyzer model ETY-96 (Toyo Sokki ■). This absorbance is proportional to the number of surviving cells. TN
Calculate the cell growth rate from the absorbance with the absorbance of the control without F added as 100% and the absorbance of the blank with no cells as 0%, and create a cell growth rate curve (horizontal axis: TNF administration/vertical axis: cell growth rate) do. 5 of the cell growth rate of the control without TNF.
The concentration of TNF corresponding to the 0% value was calculated from the regression equation of this curve, and was defined as the 50% effective dose (hereinafter abbreviated as ED50). Hereinafter, the effect of enhancing the in VltrO antitumor action of TNF in the present invention will be evaluated based on this comparison with EDSO.

A comparison with the case of single administration is shown in Figure 1.

The ED50 of the control (administered rl-tu-TNF alone) was 600 LJ/d, whereas the EDso in the case of co-administration of rHu-TNF/trypsin was 200 U/d. rHu-TNF
It is clear that the sensitivity to

<Example 2> Antitumor action enhancement effect of rf-1u-TNF by trypsin The cytotoxic action of trypsin (Hawai/Osaka University Research Institute) on Noshi929 cells was enhanced by the above-mentioned rf-1u-TNFin Vi.
As a result of testing according to the trO assay method, EDSO was 313 U/d, and the highest dose that did not show cytotoxic effects was 1.
It was 56U/-.

rHu-T with a trypsin administration concentration of 150 U/tolerance
The changes in the cytotoxic effect on L929 cells when the NF administration concentration was changed from 10 to 10" U/d (serial 2-fold dilution in 11 steps) were investigated. The cytotoxic effect of papain (Merk) on L929 cells was assayed in the same manner as in Example 1. As a result, EDso was 3.
．． The highest dose that showed no cytotoxic effect was 1.95 μg/mfl of 91 μg/− or more.

FIG. 2 shows the results of examining the effect of papain on enhancing the antitumor effect of rHu-TNF on L929 cells in the same manner as in Example 1, with the papain administration concentration being constant at 2 μg/d.

Control (rHu-TNF alone administration) EDSO was 600 U.
/-, whereas the EDso in the case of rHLI-TNF/papain combined administration was 300 U/d, which clearly shows that Tokara and Hahainha L929 cells are sensitized to rHu-TNF.

[Brief explanation of drawings]

In Figures 1 and 2, the horizontal axis represents TN Fll1 degree, the vertical axis represents L929 cell growth rate, and the plot marked O is the control (
TNF alone administration), plots marked with * indicate combined administration. It is a graph showing the correlation between TNF administration ■ and L929 cell growth rate.

Claims (2)

[Claims] (1) An antitumor pharmaceutical composition containing at least a tumor necrosis factor (component A) and a protease (component B) as active ingredients. (2) The composition according to claim 1, wherein the protease is at least one selected from the group consisting of trypsin and papain.