JPH0381223A - Tnf inducer - Google Patents

Abstract

PURPOSE:To provide a TNF inducer capable of inducing TNF alpha having highly necrotic effect on tumor, containing, as active ingredient, a camptothencin or its derivative. CONSTITUTION:The objective medicine containing, as active ingredient, a camptothencin of the formula {R1 is H, Cl or 1-4C lower alkyl; R2 is H or -OCO-X [X is Cl or -NR3R4 (R3 and R4 are each H or alkyl, or forming heterocycle by mutually binding together with N)]} or its derivative. The present medicine inhibits DNA topoisomerase I, inducing the gene manifestation of TNFalpha (human tumor necrosis factor) and producing active-type TNFalpha. The TNF alpha has also leukemia cell-differentiating and endotoxin-like phlogogenic effects. A compound of the formula is e.g. 9-(4-(isopropylcarbamoylmethyl)-1- piperazino)carbonyloxycamptothencin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a TNF inducer that induces TNFα, which has a strong necrotic effect on tumors.

[Prior Art] Recently, in the field of cancer treatment, BRM (biolo
logical rasponsa modifiers
) concept has come into the spotlight. BRM refers to substances for treating cancer by modifying the host's biological responsiveness to tumors. Immunotherapy agents are commonly associated with this. Immunotherapeutic agents can be understood to have almost the same meaning as BRM, but strictly speaking, immunotherapeutic agents strengthen the host's immunological surveillance mechanism against cancer.
As a result, cancer treatment is performed. The difference between immunotherapy and the BRMII method is that in immunotherapy, the immunotherapeutic agent itself does not have direct antitumor activity, but BRM itself has antitumor activity.

Currently, as it has become clear that the therapeutic effects of interferon (hereinafter abbreviated as IFN) are limited to specific patients, interleukin-2 and other lymphokines are attracting the most attention instead of TFN. There is TNF.

Tumor necrosis factor (
TNF, named tumor necrosis factor, is one of the lymphokines discovered in 1975.

Human TNF has a molecular weight of 70. Goo, low or high DH
Although it is inactivated when treated for 12 hours at pH 6 to 8, it is stable against heat when treated at 56°C for 60 minutes, and is inactivated at 70°C for 60 minutes. It has been known. Recently, using genetic recombination technology, human TN
F. DNA was cloned and the entire base sequence of human TNF was determined (Pennica, D.

at al., Nature, 312, 724 (198
4)) According to this study, it was revealed that TNF consists of 156 amino acids and has a similar gene sequence to hydrinphotoxin, a factor that exhibits cytotoxicity to cultured cells. When the molecular weight is calculated from this result, it is estimated to be 17,358, and from the molecular weight of natural TNF, it is thought that the TNF molecule is probably a multimer. Furthermore, currently this human TN
F is sometimes referred to as TNFa, and hydrinphotoxin is sometimes referred to as TNFa.

TNFa, which has non-species-specific characteristics unlike IFN
T is cytotoxic to tumor cells in vitro, and normal cells are resistant to TNFa.
There are cells that are sensitive and cells that are not sensitive to NFa. For example, L-929 cells are highly sensitive to TNFa and are a cultured cell line used for TNF titer measurements.

The mechanism of action of TNFa is not yet well understood, but its main action is thought to be through direct action on tumor cells that are actively engaged in metabolism. Others have been reported to enhance RNA synthesis (Ostrove et al.
,J.M., ,Gifford, G.E., ,Proc.
Soc.

Exp, Biol, Med, 160,354 (197
9)) That is, it has been shown that when a metabolic inhibitor and TNFa are used in combination with L-929 cells, the necrotic effect of TNFa is enhanced, and when TNFa acts on cells, the cells resist the damaging effects of TNFa. , compensatory RNA
This is interpreted as increasing synthesis. In addition, a report examining the influence of TNFa on the cell cycle (Darzynki et al.
ewicx.

Z. et al., Cancer Res, 44.
83 (1984)'), it was found that when TNFa was applied to L cells, the cell cycle was arrested at the 02 stage and had no effect on the 61st or 8th stages. TNFa-induced cell damage probably occurs late in the mitotic phase or immediately after cytokinesis;
It is thought that it does not affect DNA synthesis or protein synthesis.

On the other hand, camptotensin (hereinafter abbreviated as CPT) is
Camptothpca acumin
It is an alkaloid extracted and isolated from plants such as Nyssacaae) and has a strong nucleic acid synthesis inhibitory effect.The action is rapid and reversible, and has a unique effect of not exhibiting cross-resistance with existing anticancer drugs. It is an antitumor substance with a mechanism. In other words, its mechanism of action is to use DNA boisomerase I ()-Boi) as an intracellular target.
- By immobilizing topo I complexes, O
N^ It inhibits metabolism and causes cell death.

Furthermore, various attempts have been made to obtain CPT derivatives with improved pharmacological activity and toxicity by chemically modifying natural CPT (Japanese Patent Application Laid-open Nos. 19790-1979 and 85319-1980). ).

[Problems to be Solved by the Invention] When we were investigating the mechanism of action of CPT and its derivatives during transcription in tumor cell genes, we discovered that CPT and its derivatives not only suppress transcription but also exert novel effects on several genes. It was found that expression was induced.

Furthermore, as a result of intensive efforts, the present inventors analyzed the expressed genes and found that one of them was the TNFα gene, and demonstrated that ZeroPT and its derivatives have good TNF-inducing ability. This confirmation led to the present invention.

Furthermore, the present inventors have discovered that RP, which is a cultured cell derived from human acute T-lymphocytic leukemia cells (T-ALL),
The present invention was achieved by confirming that when MI8402 cells are treated with CPT and its derivatives, TNFα mRNA is specifically induced and synthesized, and is released into the culture medium as an active TNFα protein. It is.

[Means for Solving the Problems] The TlrF inducer according to the present invention contains CPT and its derivatives represented by the following structure as active ingredients.

(However, in the formula, R1 is a hydrogen atom, a chlorine atom, or a lower alkyl group having 1 to 4 carbon atoms.

R2 is a hydrogen atom or a group represented by the formula -oco-x. This R2 is the 9th and 10th position of the camptotensin skeleton.
It may be located in any of the 1st, 11th, and 12th positions.

This X is a chlorine atom or a group represented by the formula -NR3R4. Rs and Ra are each a hydrogen atom or a substituted or unsubstituted alkyl group, and R3゜R4 may be taken together to form a heterocycle with the N atom to which they are bonded, and the heterocycle The ring may contain a heteroatom in addition to the N atom) Specific derivatives include 9-(4-(isopropylcarbamoylmethyl)-1-piverazino)carbonyloxycamptotensin, 1O-(4-(isopropylcarbamoylmethyl)-1-piverazino), 1.1O-(1-piperidino)-1-piperidino)carbonyloxycamptotensin, 1l-(4-(isopropylcarbamoylmethyl)-3-piperazino)-carbo Niloxycamptotensin, 1l-(4-ethyl-1-piperazin carbonyloxycamptotensin, 12-(4-(1-piperidino)-1-piperazin carbonyloxycamptotensin, 12-(4-(isopropyl) carbamoylmethyl)-l-piperazino)carbonyloxycamptotensin, 7-methyl-9-(4-(isopropylcarbomoylmethyl)-1-piperazinocarbonyloxycamptotensin, 7-nityl-9-(4-(1 -piperidino)-1-piperisivacarbonyloxycamptotensin, 7-nithyl-1O-(4-(isopropylcarbamoylmethyl)-1-piperazino)carbonyloxycamptotensin, 7-nithyl-1O-(4-(1- piperidino)-1-piperidino) carbonyloxycamptotensin (abbreviation cPT-1l), 7-nithil-1O-(1-biberazihacarbonyloxycamptotensin, 7-nithil-1O-(4-propyl-1-piperazino) ) Carbonyloxycamptotensin, 1O-(4
-benzyl-1-piperazino)-carbonyloxy-7-
Ethylcamptotensin, 7-Nithyl-1O-(4
-(3-hydroxypropyl)-1-piperazino)carbonyloxycamptotensin, 7-nityl-1O-
(N-methyl-N-(2-dimethyl-7minoethyl))aminocarbonyloxycamptotensin, 7-nityl-1O-(N-methyl-N-(1-methyl-4-piperidino)amivarcarbonyloxycamptotensin ,1
O-(1=(4-N,N-dimethylamihabyperidino)
carbonyloxy-7-ethylcamptotensin,
1O-(1-(4-N,N-diethylamino)piperidino)carbonyloxy-7-ethylcamptotensin,
10- (1-(4-N, N-di-herb 10- (1
-(4-N,N-di-n-propylamino)piperidino)carbonyloxy-7-ethylcamptotensin, 1
O-(1-(4-N,N-di-n-butyl7mihapiperidihacarponi0xy-ethylcamptotensin, 7
-Nichilu 10- (1-(4-(1-pyrrolidino))
piperidino) carbonyloxycamptotensin, 7-
Nichiru 1O-(4-(1-morpholino))piperidino)carbonyloxycamptotensin, 7-Nichiru 1l-(4-(1-piperidino-1-piperidino)piperidino)carbonyloxycamptotensin, 7-Nichiru 12- (
4-(1-piperidino)-1-piperazin carbonyloxycamptotensin, 7-nithil-12-(4-(
isopropylcarbamoylmethyl)-1-piperazinocarbonyloxycamptotensin, 9-(1-piperazino)carbosiloxy-7-proylcamptotensin, 1(1:(4-(isopropylcarbamoylmethyl)-1-piperazino)- Carbonyloxy-7-probylcamptotensin, 7-butyl-12-(4-
(1-piperidino)-1-piperidino)carbonyloxycamptotensin, 7-butyl-12-(4-dimethyl7mino-1-piperidino)carbonyloxycamptotensin, 7-ri-a-10-(4-(1- (piperidino) carbonyloxycamptotensin, lO-chlorocarbonyloxy-7-ethylcamptotensin, 10-chlorocarbonyloxycamptotensin, 1O-7minocarbonyloxy7-
Ethylcamptotensin, 1O-(2-diethyl7mihaethyl7minocarbonyloxy-7-ethylcamptotensin, 1O-diethylaminocarbonyloxy-7-
Ethylcamptotensin, 7-Nithyl-1O-(1
-morpholino) carbonyloxycamptotensin,
7-Nityl-1O-(1-piperazino)carbonyloxycamptotensin, 7-Nityl-1O-(4-methyl-1-piperazino)carbonyloxycamptotensin 1.7-Nityl-1O-(4-ethyl-1-piperazino) ) Carbonyloxycamptotensin, 7-Nithyl-1O-(4-(p-methoxyphenyl)-1-piperazino) Cabronyloxycamptotensin, 7-Nithyl-10-(4-(3-hydroxypropyl)-1-piperazino) ) carbonyloxycamptotensin, 7-
Nithyl-1O-(4-(isopropylcarbamoylmethyl)-1-piperazino)carbonyloxycamptotensin, 7-totyru-1O-N-methyl-N-(1-methyl-4-piperidino)aminocarbonyloxycamptotensin, 1O -(1-morpholino)carbonyloxycamptotensin, 1O-(4-methyl-1-piperazinocarbonyloxycamptotensin, 7-nityl-1O-(4-propyl-■-piperazino)carbonyloxycamptotensin, etc. The specific structures of the above derivatives are shown in Table 1 below.

[Function] In the present invention, CPT and its derivatives are used as active ingredients.

CPT, which has an inhibitory effect on topoisomerase I (topo I) in mammals, is an intermediate in the enzyme action.
By accumulating the A-topo I covalent complex, D
It is known to be an antitumor plant alkaloid that inhibits the synthesis of NA and RNA.

In this example, genes whose transcription was induced as a result of topo I inhibition by CPT were analyzed by the Northen plot method, and it was found that TNFa was one of them.

(1) TNFa-m in human ALL cells by CPT
RNA was induced and synergistically enhanced by phorbol ester (hereinafter abbreviated as TPA), which is known to induce TNFa expression in HL-60 cells and the like. Its induction was inhibited by actinomycin D and was not enhanced by cycloheximide. It was also not cell cycle dependent.

On the other hand, in the CPT-resistant strain CPT-ni-5, no induction was observed.

(2) It was suggested that a kinase other than protein kinase C was involved in the induction of expression.

(3) By CPT and its derivative CP T-11, T
NFα activity was released during culture, and the activity was neutralized with a TNFα monoclonal antibody. The molecular weight of TNFa is
By immunoplotting, it was found to be the same as standard TNFα at 17 d.

From the above, CPT and its derivatives induce the gene expression of TNFa through topo I inhibition, and the active form of TNF
It was found that α was produced.

That is, at relatively low concentrations of CPT and its derivatives, TPA
When used in combination with TPA, TNFa was induced to be synthesized synergistically.

TNFa is a monokine mainly produced by macrophages, which are one of the body's immune cells, and has various physiological activities such as antitumor activity, differentiation of leukemic cells, and endoxin-stimulating inflammatory activity. B has such a variety of effects.
RM (biological response)
dafiers) is induced by CPT and its derivatives.When this drug is administered to a living body, it not only directly attacks tumor cells but also exerts antitumor activity through the induction of TNFα and other lymphokines. In this study, it was observed in cultured leukemia cells that CPT and its derivatives induce TNFα even when administered to living organisms.
It has great potential to become a useful anticancer agent that has a synergistic effect with promoters such as TPA.

[Example] Examples leading to the present invention will be shown below. In an example,
TPA, actinomycin D, cycloheximide, MO
PS is manufactured by Sigma, guanidine thiocyanate is manufactured by Fluka, H7 is manufactured by Seikagaku Corporation, and other reagents are manufactured by Wako Pure Chemical.
I used a company-made one. CPT and its derivatives were manufactured by Yakult Honsha, and staurosporine was manufactured by Kyowa Hakko ■. In addition, TPA, CPT, and staurosporine are used at a concentration of 1
It was used after being dissolved in DMSO at a concentration of 00 times.

Example-1 (Expression of TNFα by CPTjL treatment and synergistic expression of TNFα by combined use of CPT and TPA) [Cells and culture] Human T-cell acute lymphocytic leukemia cells (T-ALL)
RPM18402, a cultured cell derived from RPM18402, and its CPT
For the resistant strain CPT-5, fetal bovine serum was added at a concentration of 10% to RPM11640 medium (10.4 g manufactured by Hinaga Seiyaku ■, 1.0 g of baking soda, 60 a+g of penicillin G-potassium added, distilled water iIL, filter sterilized). The cells were cultured in a medium and subcultured at intervals of three cells.

[Preparation of RNA] At a cell number of 5 x 10' cells/ml, after treatment with each drug for each time, 5 x 10' cells were washed with PBS, and total RNA was purified by Pitor-Komczynski (:h
The method of Chomzynski et al.
P,and 5acchi,N,,Anal,Bioc
hem, 162158-159 (1987)). The cells were pelleted with 750 μA of 5alnD (4 M guanidine thiocyanate, 25 mM Na citrate (p
H7,0), 0.5% N-laurolisarcosine, 0.
After dissolving in 1M2-mercaptoethanol), 75μJ2
Add 2M Na acetate (pH 4.0), hydrated phenol at 750μ℃, and chloroform:isoamyl alcohol (49:1) at 150μ℃, stir, and dilute in water for 15 minutes.
Leave it for a minute. x After centrifugation at 10,000g for 20 minutes
NA was collected. After dissolving the RNA again in 5olnD, precipitating with isopropanol and washing with 75% ethanol, total RNA was dissolved in 0.1% SDS, 25mM E D T.
A, (9Ha, o) was dissolved and used as a sample.

[Gel electrophoresis method] Agarose ME (Gyudon Kagaku) Ig and sterile distilled water 741
111 was added and autoclaved at 120°C for 3 minutes to dissolve. After cooling the agarose gel to 60t,
01177) 10x MOPS Hy77-F (200
mM OPS Na (pH 7,0), 50++
+1 Na acetate, 10+ aM ED TA) followed by 16.5+ aM of 37% formaldehyde (final concentration 2.2 M), and the agarose gel was poured into a horizontal electrophoresis apparatus. Total RNA is 15 per 9.3μ square
μg, 20 μl of mixed bed.
Resin (mixed bad resin)' AG5
Formamide deionized with 01-X8(D) (manufactured by PyroRad) was added and mixed (final concentration 50%).

Then 6.7 μl of deionized 37% formaldehyde (
1Ox buffer F (final concentration 2.2M) and 4 μC was added and mixed. 10.Heated at 60℃ for 5 minutes and immediately cooled on ice.
IIA sample buffer (20% Ficoll, 20%
mME DTA, ethidium bromine 100
μg/if) was added to the gel. The gel was covered with plastic wrap and electrophoresis was performed at a constant voltage of 50 V at room temperature for 8 hours.

[Nogin blotting] After electrophoresis, transfer the gel to 25011177) 20X S S C
(x I S SC; 0.15M NaC1,0,0
Soak in 1514 Na citrate for 30 minutes, then re-incubate with 20X
Soaked in 100 ml of S S C for 10 minutes. On the transfer device core, two sheets of Whatman 3MM Vapor (manufactured by Whatman), the gel, Hybond N (manufactured by Amarjam), and two sheets of Whatman 3MM Vapor are placed in order from the bottom, and then vapor towels are layered to a thickness of about 15 cm. Cover the area with plastic wrap, layer vapor towels to a thickness of about 15 cm, place a glass plate on top of that, and then put about 1 kg on top of that.
The weight was placed on the ship, and the transfer was made at night. After drying Hybond N,
i+) with the RNA-attached side of the filter facing down, and was irradiated for 5 minutes and fixed.

[Hybridization] The Hybond N filter with immobilized RNA was prepared using 50% Form 71, 5xSSPE (1xSSPE; 0.1
8m NaCl 1,0,01M Na phosphate (pH7,7
), 0.001MEDTA), 5X Denhard (De
nhardt) solution (100x concentration solution: 2% BSA,
2% Ficoll, 2% polyvinylpyrrolidone), 0
．． 4 with 1% SO3, 20ag/ml heat-denatured E. coli DN^
Prehybridization was performed at 2°C for 4 hours. Next, add dextran and random primer to a final concentration of 10% in new hybridization buffer.
With the labeling kit (manufactured by Takara Shuzo), “P-d”
CTP (manufactured by Amajam Co., Ltd. 3 (1 (lOci/mol)
Pst containing the fourth exon of the TNFa gene labeled with
I/EcoRl 850bp fragment (s
x to' cpra/u g) at 42°C.
Hybridization was carried out for 6 hours.

Then at 60°C, 2x SSPE, 0.1% SDS for 15
After washing twice for 2 minutes, 1x SSPE, 0.1% S at the same temperature.
Washed with DS for 30 minutes. Furthermore, 0. lx S S P
E, Washed with 0.1% SDS at room temperature, Kodak XA
Exposure was carried out using R-5 film (manufactured by Kodak) with a Cornex intensifying screen at -80°C.

Through the above operations, the parent strain RPMI8402 (a) and the CPT-resistant strain CPT- were treated with the T.
We looked at temporal changes in NFα gene expression.

Figures 1a and 1b are explanatory diagrams showing the results of using CPT%TPA alone and in combination.

In the figure, lanes 1 to 6 are 30 nM TPA,
Lanes 7-12 are 30 nMT P A + 4 μM
CP T , lanes 13-18 are 4aMCPT, lanes 1, 7.13 are drug! A 0 hours, lane 2
, 8.14 is drug treatment for 30 minutes, lane 3, 9.15 is drug IA treatment for 1 hour, lane 4, 10.16 is drug treatment for 2 hours, lane 5.11.17 is drug treatment for 4 hours, lane 6
, 12.18 schematically shows the results of preparing total RNA after 8 hours of drug treatment and analyzing it by the Nogin Butting method. Note that the arrow indicates the size of TNFα-mRNA.

Human T-cell acute lymphocytic leukemia cells (T-ALL)
In cultured cells derived from RPMI8402, TPA ('phorbol ester), which is known to express TNFα (Fig. 1a, lanes 1 to 6), and the DN8-voisomerase I-specific inhibitor CPT (Fig. lane 1
3-ta) The expression of the TNFα gene was observed to the same extent by the Nogin plot method. Furthermore, when these drugs were used in combination at the same concentration (lanes 7 to 12 in Figure 1a), synergistic expression induction was observed that reached a peak 4 hours after addition (lane 11 in Figure 1a).

On the other hand, the highly resistant CPT strain CPT-
In 5, no expression induction by CPT was observed (
(Fig. 1b, lanes 13 to 18), TNFα expression was observed only with TPA (Fig. 1b, lanes 1 to 6), and expression by C in combination with TPA (Fig. 1b, lanes 7 to 12) was observed only with TPA.
6 or more, which was approximately the same amount as the car-treated treatment, it was considered that CPT induced TNFα-a+RNA through topoⅠ inhibition.

Example 2 (Inhibition of TNFα-mRNA expression induction by CPT with actinomycin D) Whether the induction of TNFα-mRNA expression by CPT is mediated by RNA synthesis or not was determined using actinomycin D (hereinafter referred to as an RNA synthesis agent). , AMD).

FIG. 2 is an explanatory diagram showing the results of inhibition of TNFa-mRNA expression induction by AMC by CPT. In the figure, lanes 1 to 5 were treated with 30 nMT PA for 45 minutes, and lanes 6 to 1O were treated with 30 nMT PA. P A +4 u
MCPT and lanes 11-15 are 4aMCPT and 4
showed inhibition of TNFα-mRNA synthesis induced by AMD upon treatment with time. Lane 2, 7.12 is o,
ooau g/ml, lane 3, 8.13 is 0.0
4ag/ml, lane 4.9.14 is 0.2μg
/+1. Lane 5. 10. 15 is 1μg/la
1 of AMD was added 5 minutes before induction. After the treatment, total RN^ was prepared, Nogin blotting was performed, and the analysis results are schematically shown.

Figure 2 lane 1 is TPA alone, Figure 2 lane 6 is TPA
, lane 11 in FIG. 2 shows expression induction by treatment with CPT alone, and lanes 2 to 5 in FIG. Lane 7-1
0. Lanes 12 to 14 are TPA cars, CPT cars,
Various concentrations of AMD were added during treatment with TPA and CPT alone.

In both cases, the amount of TNFα-mRNA synthesized was
decreased in a concentration-dependent manner, and was completely inhibited at 1°C g/ml AMD (lanes 5.10.15 in Figure 2). The above indicates that the induction of TNFα-mRNA by CPT was expressed by inducing new transcription.

Example 3 (Enhancement effect of cycloheximide on CPTe-mediated induction of TNFα-mRNA expression) Protein synthesis inhibitor cycloheximide (hereinafter referred to as CHX)
We examined the presence or absence of suba-induction due to (abbreviated as).

FIG. 3 is an explanatory diagram showing the results of the enhancement effect of CHX on the induction of TNFa-mRNA expression by CPT,
In the figure, lane 1.2 is 30 nMTPA.

30 min, lanes 4 and 5 30 nM T P A +
4 pM CPT, 4 hours, lane 7.8 4°C MC
PT, treated for 4 hours. Lanes 2 and 5.8 were added at a concentration of CHXIO μg/ml 30 minutes before the various drug treatments described above. 3 for 60 minutes and lane 6 for 4 minutes as a control.
The sample was treated with CHX alone for 30 minutes. Total RN^ was prepared from each cell, and TNF was detected by nogin blotting.
The results of analyzing the α expression level are schematically shown.

No induction of TNFα-mRNA was observed by CHX treatment alone (lanes 3 and 6 in Figure 3). When induced by TPA, slight enhancement by CHX was observed (lane 2 in Figure 3). Combination of TPA and CPT and T by CPT
NFα-mRNA induction was not enhanced by CHX (Fig. 3, lane 5°8).

The above can be achieved by using the protein synthesis inhibitor CHX.
As a result of the decrease in unstable proteins that promote the destabilization of Fα-mRNA, it was strongly suggested that TNFα-mRNA was not accumulated. Furthermore, considering the results of AMD treatment, it was considered that TNFα-mRNA transcription was induced by CPT.

Example 4 (Changes in the cell cycle of TNFα-mRNA expression induced by CPT) In order to investigate the cell cycle dependence of TNFα-mRNA expression by CPT, cells were synchronized to the G,/S boundary using the excess tykudine method. (Figure 4 a-1).

[Synchronization to the G I/S boundary] Synchronization of cultured cells occurs when cells in the logarithmic growth phase
After 16 hours of thymidine treatment, 1
Grow for 5 hours, add 1 a+M thymidine and add 1
Culture was carried out for 6 hours, and synchronization was achieved near the G,/S boundary of the cell cycle. The degree of synchrony was confirmed by flow cytometry.

[Flow cytometry method] Flow cytometry is performed by Krishan
) and other methods (Kr1shan, A, :J, cell, B
iol, 66.188-193 (1975)). Each cell population (lx10' cells) is P B
After washing with 10 nl of S, 70% ethanol at 4° C. was added to the plate while stirring for fixation. Fixed cells were diluted with PBS for 1 m
The mixture was incubated at 37° C. for 30 minutes with RNase at a concentration of g/ml. Next, after washing the cells with distilled water, the DNA was extracted with Providium iodide (0.05% with 0.1% sodium citrate).
mg/ml). Stained cells are E
Analysis was performed using a PIcs II (Coulter) cell sorter.

Figures 4a and b show TNFa-mRNA by CPT.
FIG. 2 is an explanatory diagram showing the results of changes in the cell cycle due to expression induction; in Figure a, 1 is Gd
This is a flow cytometry pattern of cells synchronized with the S boundary and immediately after removal by washing with Tiyodine.

2.5 showed the pattern after 4 hours of washing and 16 hours of culture. 4 and 7 are 4℃ MCPT & 3 at the time of 2.5.
6 is a pattern treated with 0.1% DMSO for 4 hours as a control. In Figure b, lane 1 is 4aMCPT of logarithmically growing cells and lane 2 is 0.1% D.
MS0, total RNA treated for 4 hours, lane 3.4 is a-3
, 4 total RNA at each time point, lane 5.6 is a-6,7
T by nogin proteining of all RN^ at each time point of
The results of analysis of the expression level of NFα are schematically shown.

4 hours after removal of thymidine by washing, most of the cells were in the early stage 5 (Figure 4 a-2) and 16 hours later, most of the cells were in stage 03 (stage 4
At the time shown in Figure a-5>, each sample was treated with CPT for 4 hours, and TNFα-mRNA induction was examined. b) The figure is a nogin blotting. Figure 4b lane 1.
2 shows the logarithmic growth phase, lanes 3.4 in FIG. 4b show the 5th phase, and lanes 5.6 in FIG. Figure 4b Lanes 4 and 6 are DMSO1 as control.
Lanes 1 and 3.5 in FIG. 4b were treated with 4 μM CPT for 4 hours. TNFα-mR in both 5th and G1 stages
NA induction was observed.

As described above, no cell cycle dependence was observed in the induction of TNFα-mRNA by CPT.

Example-5 (in the presence of protein kinase inhibitor, C
Inhibition of TNFa-mRNA induction by PT) TPA inhibits protein trade kinase C-kinase (hereinafter referred to as
It is known that the TNFα gene is activated through the activation of protein kinase C (abbreviated as PK-C). Therefore, TNFα in combination with CPT or TPA
We investigated whether PK is also involved in the transcriptional induction of .

Figures 5a and 5b are explanatory diagrams showing the results of inhibition of TNFα-mRNA induction by CPT in the presence of a protein kinase inhibitor; in the figures, a is H7IA;
b is staurosporine treatment.

In Figure a, lanes 1 to 5 and lanes 6 to 10 are 4 μM with 30 nMTPA + 4 μM CPT and 4aMCPT, respectively.
Expression of TNFα-mRNA upon time treatment. Lanes 1 and 6 are 0 μM1, Lane 2.7 is 5aM1, Lanes 3 and 8 are 10 μM1, and Lane 4.9 is 20 μM. 40 μM H7 was added 30 minutes before adding CPT and other inducers. After preparing gold RNA after treatment, the expression was analyzed using the Northern blotting method, and the results are schematically shown.

In figure b, lanes 1 to 7 and lanes 8 to 14 are 30 nM TP A + 4 μM CP T and 4a, respectively.
This is the expression of TNFα-mRNA upon treatment with MCPT for 4 hours, lanes 1 and 8 are Ong/ml, lanes 2,
9 is 0.5ag/ml lane 3. lO is 1 ng
/ml Lane 4.11 is 2ag/ml, Lane 5.12 is 4ag/ml, Lane 6.13 is 8a
g/ml, lane 7.14 is 16 ng/I! 11 of staurosporine was added before inducer treatment.

Total RNA was prepared from treated cells and the expression level of TNFα was analyzed by nogin blotting. We investigated whether staurosporine, an inhibitor specific to PK-C, blocks the induction of TNFα.

Figure 5a shows the results for H7, and Figure 5b shows the results for staurosporine. Figure 5a lanes 1-5. Figure 5b lanes 1 to 7 are TPA and CPT combined, Figure 5a lanes 6 to lO1 5b
Lanes 8 to 10 in the figure show induction by CPT treatment alone.

When treated with protein kinase inhibitor H7 in CPT and in combination, TNFα was inhibited at low concentrations of 5 to 10 μM.
-inhibited mRNA induction (Fig. 5a, lanes 2 to 5.7)
~10). The cytotoxicity of H7 was higher than that of drug-untreated RPMI84.
When the proliferation of 02 is taken as 100%, the concentration that inhibits proliferation by 50%, that is, ICs. was 17 μM. On the other hand, PK-
Staurosporine, an inhibitor of C, is in both cases fan
There was no inhibition up to g/ml. Staurosporine cytotoxicity Ias. was 4ag·/all. From the above, it was suggested that TNFα-mRNA induction by CPT alone or in combination involves activation of protein kinases other than PK-C.

Example-6 (L-29 in the culture supernatant of CPTf1 rational cells)
Cytotoxic activity against 9 and neutralization by human TNFα monoclonal antibody) We have shown above that TNFα-mRNA is transcriptionally induced by CPT;
It was investigated whether TNFα with translated activity was produced. As a derivative of CTP, among the derivatives with high antitumor effects, 7-nityl-1O-(4-(1-piperidino)-1-biperidihacarponyloxycamptotensin (4th name cp T-11) was used. .

[Induction of TNFα activity] RMI8402 in logarithmic growth phase was added to RPM11640 medium (
Wash with serum-incompatible medium and incubate 2 x 10' cells with the same medium (serum-incompatible).
cells/ml. X 1000 times more concentrated TPA
, CPT-11 and TPA, CPT-11 mixed solution in 1/
1000 was added and cultured at 37°C, and the culture supernatant was used as a sample. The sample was dialyzed against PBS and TN
Fα activity was measured. In addition, if necessary, the sample was concentrated 20 times using CentriBleb (Amicon).

[TNFα activity measurement method] TNFa activity measurement method is carried out using Carswell (Carswell).
) and other methods (Carswell, E, ^, :Pr
o, Nat1. Acad, Ll, S, A.

72.3686-3670 (1975)) was used with some modifications. The final method takes advantage of the adhesive properties of L-929 cells, removes cells that have been damaged by TNFα and have lost their adhesive properties by washing, stains the remaining viable cells, and measures the staining concentration. This is a method of measuring loss ratio. Target a cell L-
929 cells 4 x 10' cells/100μ
The cells were placed on a 96-well microtiter plate and cultured for 4 hours in a 37°C CO2 incubator to allow cells to adhere. Subsequently, 50 μl of diluted sample and 4
μg/+nl actinomycin D (AMD) solution 50
μm (final concentration 1 μg/ml) was added and cultured at 37° C. for 18 hours. After washing each well with PBS, 0.
After staining with 1% crystal violet, 1% MeOH in PBS for 20 minutes, the plate was washed with tap water and 0.
It was dissolved in 100 μl of 1% 5DS and the absorbance at 570 nm was measured. The survival rate of the TNFα activity was calculated from the ratio of the absorbance of the control to which no sample was added.

FIG. 6 is a diagram showing the cytotoxic activity against L-299 in the culture supernatant of CPT-treated cells, and FIG.
FIG. 2 is a diagram showing the results of neutralization with a monoclonal antibody against NFα.

In FIG. 6, the vertical axis represents L-9 of the drug-untreated culture supernatant.
The cytotoxic activity of the treated culture supernatant is expressed as a survival rate when the cell survival rate against No. 29 is taken as 100%. The horizontal axis indicates the processing time after addition of the drug.・ is 4μMCPT, mu is 30n
MT P A , ■ is 30 nMT P A + 4 μ
Indicates MCPT.

In Figure 7, the vertical axis is L-92 of the drug-untreated culture supernatant.
The cell survival rate for 9 was set as 100%. The horizontal axis is TNF
0 mAb, which indicates the dilution factor of α monoclonal antibody (hereinafter abbreviated as mAb), was added at the same time as the culture supernatant, and the determination was made 18 hours later.・ indicates untreated culture supernatant, ○ indicates TPAIA culture supernatant, and MU indicates CPTlf! 1 Physical culture supernatant, △ TPA
+CPTIA culture supernatant, ■ indicates human rTNFα.

Each point on the right side of the figure showed cytotoxic activity in the absence of mAb.

Cytotoxic activity was induced by TPA, CPTJIL, and their combination, appeared in the culture supernatant after drug treatment, increased over time, and reached a plateau at 8 hours (Figure 6). The activity induction was correlated with the amount of TNFα-mRNA. On the other hand, the activity could not be detected using DMSO alone as a control.

Furthermore, the cytotoxic activity induced by TPA, CPT, and their combination was inhibited by a human TNFα monoclonal antibody (mAb) in a concentration-dependent manner, and was completely neutralized at a high concentration (FIG. 7).

Example-7 (Identification of TNFα molecules induced by TPA, CPT (-11) alone and in combination by immunoblotting) [Identification of TNFα by immunoblotting] Each drug
After inducing NFα, protein quantification of the culture supernatant was performed using a Bio-Rad protein assay kit using γ-globulin as a standard.

That is, take culture medium equivalent to 100 μg of total protein and bacterial mass, and add 100% trichloroacetic acid to a final concentration of 10%.
was added to aggregate the protein. After standing in water for 10 minutes, centrifuge at x 10,000g at 4°C.
It was precipitated. Sample buffer (540 m
M't-lis-hydrochloric acid (p)I 8.0), 50
111M dithiothreitol, 1% SDS, 7.5
% glycerol.

After adding 10 μl of 0.006% bromophenol blue and melting, heat at 100°C for 3 minutes to form 5DS-PA.
GE (10% to 20% gradient gel Daiichi Chemical) was performed. After electrophoresis, 0.1M Tris, 0.192M glycine,
It was transferred to a filter (Clearprot P membrane; ATTO Co., Ltd.) at room temperature for 90 minutes with 0.02% SDS and 20% MeOH solution at 2 mA per 1 cn'. The filter is TTB
S (1001 nM Tris-HCl (pH 7,5) 0.
After washing for 30 minutes with 9% NaCl, 0.05% Tween 20), primary serum diluted with TTBS (2.5 μs/
ml) was reacted at 4°C for 12 hours. Below, Vectastin ABC Kit Manual: Veroxidase staining method (
Vector). For color development, 100mM)-Lis-HCl (p)I 7.5) 20mg/ml in 20ml
diaminobenzidine 800 μm, 8% nickel chloride 1
00 μl and 15 μl of 3% hydrogen peroxide solution were added, and the reaction was carried out at room temperature for 15 minutes.

FIG. 8 is an explanatory diagram showing detection of TNFα in drug-treated culture supernatant by immunoblotting. In the figure, 5DS-PA
After separation by GE, TNFα molecular species were examined by immunoplot method, and the results were schematically shown. Lane 1 is human rT
NFα, lane 2 is 30 nMT P A , lane 3 is 30 nMT P A + 4 μMCPT, lane 4 is 4
μMCPT, lane 5 is 0.1% DMSO, lane 6
is 160μMCPT-11 rune 7 is 160μMC
PT-11+ 30nMT PA is shown. lane 1
is 0.4 μg as human rTNFα protein, and the total protein amount in lanes 2 to 7 is 100 μg. Right arrow indicates size marker.

The left arrow indicates TNFα standard protein trade (17,5 Kd).

CPT and its derivative CPT-11 induce TNFαm
A molecular species that immunologically crosses with Ab was induced (lane 4.6 in Figure 8). It was also synergistically induced when used in combination with TPA (lane 3.7 in Figure 8). Lane 1 in Figure 8 is standard human TNFα, with the same molecular weight of 17.5 as that of d. , a lower molecule (17Kd)
Several low-molecular-weight products that crossed with the main product and some iAbs were detected. On the other hand, low molecular weight products include protease inhibitors, transamine, FOY, aprotinin, chymostatin, antipain, leupeptin,
and to the same extent in the presence of mixtures thereof.

In the above examples, CPT and its derivatives showed that the top
As a result of the inhibition, the genes induced by transcription were analyzed by the Northen plot method, and it was found that TNFα was one of them. That is, (1) by CPT, human^
TNFα-mRN^ is induced in LL cells, and HL-6
It was synergistically enhanced by phorbol ester (hereinafter abbreviated as TPA), which is known to induce the expression of TNFα in cells such as 0 cells. Its induction was inhibited by antipain D and not enhanced by cycloheximide. It was also not cell cycle dependent. On the other hand, the CPT-resistant strain CPT
- and 5, there was no such induction.

(2) It was suggested that a kinase other than protein kinase C was involved in the induction of expression.

(3) By CPT and its derivative CP T-11, T
NFα activity was released during culture, and the activity was neutralized with a TNFα monoclonal antibody. The molecular weight of TNFα is
By immunoplotting, it was found to be 17Kd, which is the same as standard TNFα.

Based on the above, CPT and its derivatives induce TNFα gene expression through topo I inhibition, and induce active TNFα.
It was found that α was produced.

That is, at relatively low concentrations of CPT and its derivatives, TPA
to the same extent as, further &: When used in combination with TPA, it synergistically increases TNFα.
was synthesized inductively.

TNFα is a monokine mainly produced by macrophages, which are one of the body's immune cells, and has various physiological activities such as antitumor activity, differentiation of leukemia cells, and endoxin-like inflammatory activity. B has such a variety of effects.
RM (biological response m)
The fact that TNFa, which acts as an odefier, is induced by CPT and its derivatives means that when this drug is administered to a living body, it not only directly attacks tumor cells but also exerts antitumor activity through the induction of TNFa and other lymphokines. In this study, it was observed in cultured leukemia cells that CPT and its derivatives induce TNFa, even when administered to living organisms.
It has great potential to become a useful anticancer agent that has a synergistic effect with promoters such as TPA.

[Effects of the Invention] As explained above, the present invention provides camptotensin (CP
T) or its derivative is DNA topoisomerase (
By immobilizing the DNA-topo I complex using topo I complex as an intracellular target, we not only inhibit the DNA metabolism of tumor cells and cause cell death, but also induce TNFa, which has a strong necrotic effect on tumors. The ability to induce TNF was confirmed. For this reason, BRM (biol therapy) is used to treat cancer not only as a chemotherapeutic agent or immunotherapeutic agent but also by modifying the biological responsiveness of the host to the tumor.
ogtcal response 1odefiers
), it has the effect of making it possible to perform effective cancer treatment by using the powder.

[Brief explanation of drawings]

Figures 1a and b are explanatory diagrams showing the results of using CPT%TPA alone and in combination; 's2 figure is TNFa-mR by CPT.
An explanatory diagram showing the results of inhibition of NA expression induction by AMD,
Figure 3 shows CPT induction of TNFa-mRNA expression.
An explanatory diagram showing the results of the enhancement effect by HX, Figures 4a and b are explanatory diagrams showing the results of changes in the cell cycle induced by TNFa-mRNA expression by CPT, and Figures 5a and b are diagrams showing the results of changes in the cell cycle induced by CPT. Below, T by CPT at
An explanatory diagram showing the results of inhibition of NFa-mRN^ induction, Fig. 6 is a diagram showing the results of cytotoxic activity against L-929 in the culture supernatant of CPT-treated cells, and Fig. 7 is a diagram showing the results of cytotoxic activity against L-929 in the culture supernatant of CPT-treated cells.
FIG. 8 is an explanatory diagram showing the detection of TNFa in the drug-treated culture supernatant by immunoblotting.

Claims (1)

[Scope of Claims] A TNF inducer containing camptotensin (CPT) or a derivative thereof represented by the following structure as an active ingredient. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, in the formula, R_1 is a hydrogen atom, a chlorine atom, or a lower alkyl group having 1 to 4 carbon atoms. R_2 is a hydrogen atom or is represented by the formula -OCO-X This R_2 is the 9th position of the camptotensin skeleton,
It may be located at any of the 10th, 11th, and 12th positions. This X is a chlorine atom or a group represented by the formula -NR_3R_4. R_3 and R_4 are each a hydrogen atom or a substituted or unsubstituted alkyl group, and R_3,
R_4 may together form a heterocycle with the N atom to which they are bonded, and the heterocycle may contain a heteroatom in addition to the N atom)