JPS6261926A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:To provide a carcinostatic agent containing a microorganism-originated protease as an active component, exhibiting remarkable carcinostatic activity against various tumors, keeping the carcinostatic effect for a long period at the local point of the cancer and having low cytotoxicity to normal cell. CONSTITUTION:A protease originated from microorganism (e.g. neutral protease originated from Serratia marcescens) is used as an active component. The behavior of a polymeric carcinostatic agent applied locally to the tumor tissue is completely different from that of low-molecular carcinostatic agent. The polymeric substance is retained for a long period at the local tumor without being recovered and secreted either hematogenically nor lymphogenically. Accordingly, when the microorganism-originated polymeric protease is applied to the local position of cancer, an excellent carcinostatic effect can be attained and the drug action is continued for a long period. Furthermore, it has been found that the cytotocity against normal cell is considerably lower than the cytotoxity against cancer cell.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an anticancer agent containing a microorganism-derived protease as an active ingredient.

Drawing on our many years of experience in research into anticancer drugs, the present inventors have conducted research with the aim of developing effective anticancer drugs with fewer side effects. We have discovered for the first time that a protease derived from a microorganism has extremely effective anticancer activity.

The action of microbial-derived protease, which is the active ingredient of the anticancer agent of the present invention, is protein decomposition and cell destruction as a result, and it is an anticancer agent with a category 1 action that is completely different from the action of conventional anticancer drugs.

[Prior art]

The present inventors have previously discovered that the structure of blood vessels and lymph vessels in tumor tissue is significantly different from that in normal tissue. In other words, when a polymeric anticancer drug is administered locally to a tumor tissue, its behavior is completely different from that of a low-molecular substance; polymeric substances are neither recovered nor excreted via the bloodstream nor lymphatics, and remain in the tumor site for a long period of time. A phenomenon of stagnation can be seen.

This is because the large molecular weight reduces the diffusion rate of the drug and reduces its passage through the vascular endothelium.
It is believed that this decreases blood vessel penetration and subsequent recovery efficiency. Furthermore, the present inventors have already discovered that lymph vessels are absent in the tumor region. In normal tissue, the lymphatic system is the main route for recovery of macromolecules and oil droplets in these tissues, but because tumor tissue lacks this system, macromolecules such as enzymes are
It remains locally in the tumor for a long time (Iwai, Kei (I)
wai, K.) et al. Cancer Research
r Res, ) Vol. 44, pp. 2115-2121, 198
4th year, Maeda, et al., Journal of Protein Chemistry (J, Pr.
ot, Chem,) Volume 3, Pages 181-193, 1984
2013, Hiroshi Maeda, Toshimitsu Konno, “Cancer and Chemotherapy” Vol. 12, 77
3-782 1985).

The present inventors focused on these findings, and when locally injecting microbial-derived protease, which is a polymeric substance,
Some of them exert strong and long-term toxicity on cancer cells, leading to the assumption that they may have significant anticancer effects.

In the past, there were no anticancer drugs based on this idea, but recently, an anticancer drug similar to the anticancer drug intended by the present inventors has been developed that contains acidic protease derived from human urine as an active ingredient. was disclosed (Japanese Unexamined Patent Publication No. 1983)
-13523).

[Problems to be solved]

The anticancer drugs that have been developed so far are mainly low-molecular-weight ones, and even if they have excellent efficacy, they have the disadvantage of being easily diffused and therefore unable to maintain their efficacy. The anticancer drug disclosed in 1999 has as an active ingredient acidic protease present in trace amounts in human urine, but it is extremely difficult to produce this acidic protease in large quantities.

[Means for solving problems]

Therefore, the present inventors conducted extensive studies to find a drug that has excellent anticancer effects, has long-lasting efficacy, and is low in toxicity to normal cells.

We also discovered that several types of proteases derived from microorganisms have significant anticancer effects on all experimental tumors in mice. It has been shown in vitro that the administered protease maintains its action in the cancer local area for a long time, and its cytotoxicity to normal cells is considerably lower than that to cancer cells.
The present invention was completed as a result of the itro) test.

That is, the present invention relates to an anticancer agent containing a microorganism-derived protease as an active ingredient.

The microorganism-derived protease that is the active ingredient of the anticancer agent of the present invention can be prepared by culturing a microorganism and collecting it from the culture, or by purchasing a commercially available microorganism-derived protease.

When obtaining protease by culturing microorganisms, Serratia marcescens (S6 ratiamarces
cens), Bacillus subtilis
subtil, is), Streptomyces griseus, Bacillus sp, Streptomyces sp.
Microorganisms such as sp. When the culture supernatant is separated by filtration or centrifugation, protease activity is found in the culture supernatant. After concentrating the culture supernatant 2 to 20 times, it is possible to prepare purified protease with a purity of 95% or more through means such as dialysis and other steps, gel filtration, ion exchange chromatography, fractional precipitation, and salting out. can. Examples of commercially available products that can be obtained without culturing microorganisms include subtilisin and pronase (both trade names).

All of these proteases having anticancer activity are neutral proteases derived from microorganisms. However, Acrocylindrium sp.
Acidic proteases produced by sp. Its activity was completely inhibited by in vivo protease inhibitors such as macroglobulin, and no anticancer activity was observed.

Below, five production examples, test examples, and examples of the present invention are shown.

Production Example 1 Serratia-V Lucessence Kums 3958 FER
M-P No. 8436 (this strain was isolated from a human corneal ulcer patient, is a strain preserved at the Kumamoto University School of Medicine, and has been deposited at the Microtech Institute as deposit number 8436) was cultured overnight in trypto-soy liquid medium. And that 2mQ
was placed in a 1Q Sakaro flask containing 200-350+ nQ of the same medium and cultured at 30°C under shaking (1-2).After 25-30 hours when the protease activity reached its maximum equilibrium value, the culture solution was filtered. The resulting filtrate was concentrated 4 times under reduced pressure at 30°C or lower, or concentrated as a precipitate by adding 90% saturated ammonium sulfate, and then dialyzed against distilled water in a conventional manner (4°C). , 48-72 hours) and lyophilized.

This was further applied to a DEAE-cellulose column (column size, 4 x 40 cm) and eluted under a concentration gradient of NaCl using 0.0 IM Tris-HCl buffer (PH 8,3). The elution pattern is shown in FIG.

The protease activity was measured using the casein decomposition ability and the fluorescence polarization method developed by the present inventors.
, H,): Analytical Biochemistry (An
al., Biochem, ) Vol. 92, pp. 222-227, 1979.

The elution fraction can be further dialyzed and lyophilized to obtain a more purified protease using Sephadex G-100 (registered trademark) or the like (the elution pattern is shown in FIG. 2). The protease thus obtained is 0.5
Electrophoresis in a 75% polyacrylamide gel in the presence of ~1.0% Na dodecyl sulfate showed a band. The present inventors named this substance 56 protease. 56, the enzymatic chemical properties of protease are described by the present inventor and Matsumoto.
) et al. in the Journal of Bacteriology (J,
Bacteriol, ) volume 157 (1) pages 225-2
32, 1984.

Next, we investigated the cytotoxicity of various proteases to cultured cells and the influence of serum on the cytotoxicity, demonstrating that microbial-derived proteases exhibit excellent anticancer activity.

It will be explained in tests such as anticancer effect, efficacy on solid tumors in mice, and acute toxicity test.

Test Example 1 Cytotoxicity of various proteases to cultured cells and the influence of serum on the cytotoxicity: Proteases considered to be promising as anticancer agents must not be inhibited by various protease inhibitors that exist in large amounts in living organisms.

The present inventors added various proteases to various cultured cells to examine their cell-killing effects, and at the same time added serum to examine their effects.

RPMI-1640 medium containing 10% fetal bovine serum or Eagle's MEM medium 0.5+nQ
Place in an AK plastic chamber (8-hole type) and add approximately I x 104 various cancer cell suspensions (in 0.5 ml).
and culture in an incubator at 37°C in 5% CO□ (95% air composition). On the second day, one group was left as is, and O, Ln+Q protease solutions were added to the other group.
For the group, the medium was replaced with a serum-free medium and various proteases were added in the same manner, and cultured for 1 hour and then incubated for 24 hours.
By cell number and morphological examination after hours of culture.

The anticancer effect was determined.

The results are shown in Tables 1 and 2. As is clear from Table 1, the cytotoxicity of various proteases in serum-free medium is
It can be seen that it mainly appears strongly in cancer cells and hardly appears in normal cells. Furthermore, as is clear from Table 2, it was found that the enhancement of the cancer cytotoxic effects of various proteases over time differed depending on the influence of serum. That is, in 56, the cell killing effect of protease, subtilisin, and pronase was significantly increased when treated for 24 hours compared to the cell killing effect when treated for 1 hour, while trypsin and papain showed almost no enhancement. Ta.

Table 2 Changes in protease activity due to serum addition (i.e., enhancement of the effect of 24-hour treatment with serum-added protease) Cells a) Enhancement (fold) of the cytotoxic effect over time b) (Increase in response to 1-hour treatment) Sa) Aze Shin 11ep2 >64 20
8 None ~ 4 None ~ 411SG
32 16 8 2~4
2~411eLa 32 12
8 2~4 2~4C% 3
2 10 8 2 2
WISH1610842 Vero 16 10 2
, 2~4 2~411EL >4
− − 2 2GMK
2 2 2 2
2b) The numbers are the dilution times that produce the same effect as the original protease after 24 hours of treatment. For example, 32 in Table 1 indicates that the same titer is exhibited at 1/32nd the amount after 24 hours even in the presence of serum.

Test Example 2 Anticancer activity of various proteases: Efficacy in mouse solid tumors (i.e. methylcholanthrene-induced cancer (Met)
h Effect on A tumor): Mat in Ba1b/C mice
h Approximately 10 A tumors were inoculated intradermally with a syringe for 0.05 d, and after approximately 7 to 9 days, the tumor diameter was 8 to 10 mm, and each tumor was dissolved at a predetermined concentration. 0.1 mQ of each protease was injected into the tumor. Figure 3 shows the diameter of the tumor after two administrations (once each day).

As is clear from Figure 3, five of the various proteases
In No. 6, tumor disappearance was observed with respect to the four types of protease, neutral protease, pronase, and subtilisin, but it can be seen that acidic protease, trypsin, and papain have almost no tumor growth suppressive effect.

Anticancer effect of protease in Test Example 356: (1) S-
Anticancer effect of 180: S-1 produced in ddY mice in the same manner as Test Example 2
Various amounts of protease 56 were intravenously administered to 80 solid tumors for a total of 7 times every 2nd to 3rd day, and the anticancer effect was measured. The results are shown in FIG.

That is, as is clear from Figure 4, administration of 30 μg/kg or more was found to be effective, and by administering 300 μg/kg five times, the tumor almost completely disappeared, and no side effects were observed due to protease administration. There wasn't.

(2) Effect on prolonging survival of mice inoculated with Meth A tumor (ascites type): After inoculating 5 x 10 s of Meth A ascites bile carcinoma tumor cells into mice, treatment with protease was started at 56 hours after 24 hours, and treatment was carried out 10 times in total. It was administered every day. The results are shown in FIG. That is, as is clear from FIG. 5, both I B/kg and 3 mg/kg administration showed a remarkable survival prolonging effect compared to the control.

(3) Growth inhibitory effect of oral administration on Meth A solid tumor: 2 x 10' Mcth A tumor cells were injected into the flank of mice, and 24 hours later, 56
were given food containing 20 mg of protease, and the control group (1
(0 mice) were fed only food, and tumor growth was compared and examined.

When the average tumor size was compared 10 days after feeding, the diameter of the protease-administered group was 56 and 7.
7c+n, and the diameter of the control group was 8.9 cm, which clearly showed a significant difference (F test, significance level 2.5%).

Test Example 4 Acute toxicity test of various proteases: Body weight 20
After administering various proteases (56, protease, subtilisin, and pronase) dissolved in physiological saline intravenously or intraperitoneally to groups of 10 ddY mice (~25), symptoms were observed over a period of one week. No abnormalities were observed with either protease administration.

As is clear from the above, it has been found that 56 protease, subtilisin, pronase, and neutral protease produced by actinomycetes all have significant anticancer effects.

Therefore, the practical application of anticancer drugs containing these proteases as active ingredients is highly anticipated.

When these proteases are administered to humans, they can be effectively treated by direct intratumoral, intraperitoneal, or oral administration.

The dosage of these proteases depends on the size of the tumor,
Although it varies depending on the growth rate and other factors, usually 1 mg rather than 10 μg is injected into the tumor at one to several locations. For intraperitoneal tumors and other disseminated peritoneal tumors, 1 to 200 ml of diluted enzyme solution is usually injected intraperitoneally.

Furthermore, in the case of oral administration, 1 to several grams per adult is usually administered at once or in divided doses.

It can be used in the form of powder, aqueous solution, granules, capsules, enteric-coated, or oil-solubilized, and can be used as a mixture to protect against pepsin in the gastric fluid, which inactivates these proteases. Additionally, it can also be used in combination with a pepsin inhibitor.

Examples of the present invention are shown below.

Example 1 100 mg of protease 56 produced in Production Example 1 was dissolved in 10 mΩ physiological saline, filtered aseptically using a membrane filter, and the filtrate was filled into a sterilized glass container and freeze-dried. It was sealed tightly to obtain a lyophilized powder for injection.

Example 2 Protease powder 1 was added to 56 obtained according to Example 1.
00g, lactose 97g and magnesium stearate 3
After weighing and mixing them uniformly, 200 mg each of this product was filled into gelatin capsules No. 2 and 2, and an enteric coating was applied to prepare enteric-coated capsules.

Example 3 Subtilisin (trade name) 200mg was dissolved in 10ml of physiological saline, filtered aseptically using a membrane filter, and the filtrate was filled in 10mM portions into sterilized glass containers, freeze-dried, and sealed tightly. It was made into a lyophilized powder for injection.

Example 4 After weighing each of subtilisin powder Ig obtained according to Example 3, 84.5 g of crystalline cellulose, mannitol Log, 2.0 g of carboxymethyl cellulose calcium, 1.0 g of magnesium stearate, and 1.5 g of hydrogenated oil. The uniformly mixed powder was granulated using an extruder to produce granules for internal use.

Example 5 500 mg of Pronase (trade name) was dissolved in 10 mQ of physiological saline, filtered aseptically using a membrane filter, and the filtrate was filled in 10 mQ portions into sterilized glass containers, freeze-dried, and sealed. It was made into a lyophilized powder for injection.

Example 6 1 g of pronase powder obtained according to Example 5, 84.5 g of crystalline cellulose, log lactose, 2 g of calcium carboxymethylcellulose, 1 g of magnesium stearate, and 1.5 g of stearic acid were mixed well and then powdered. After producing uncoated tablets with a weight of 100 mg using a tablet machine, the tablets were coated with an enteric coating agent to produce enteric-coated tablets.

[Effects of the present invention]

The anticancer agent of the present invention containing a microorganism-derived protease as an active ingredient has the features of sustained drug efficacy and low toxicity to normal cells.

[Brief explanation of drawings]

FIG. 1 shows the DEAE-cellulose column elution pattern in the purification process of protease 56 derived from Serratia marcescens, and FIG. 2 similarly shows the elution pattern using Sephadex G-1oo. Figure 3 shows the anticancer effects of various proteases on Meth A tumors.
ff! Administered intracutaneously. FIG. 4 shows the anticancer effect of protease on S-180 solid tumor at 56. The arrow indicates the case where protease was administered at 56.
The figure shows the anticancer effect of protease on Meth A ascites adenocarcinoma tumor cells at 56, and the arrow indicates the case where protease was administered at 56.

Claims (1)

[Scope of Claims] 1. An anticancer agent containing a microorganism-derived protease as an active ingredient. 2. The anticancer agent according to claim 1, wherein the microorganism-derived protease is a neutral protease. 3. The anticancer agent according to claim 1, wherein the microorganism-derived protease is a neutral protease derived from Serratia marcescens.