JPH0761950B2 - Antitumor agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to an antitumor agent containing mycoplasma as an active ingredient, and an antitumor agent further containing interferon.

2. Description of the Related Art It is a known fact that various bacterial cells and bacterial cell components act as immunostimulators and have antitumor activity, and in recent years, studies have been conducted to treat tumors using them. For example, BCG (Bacillus Calmette Guerin) and Corynebacterium parvu
m), Streptococcus haemolytics (Streptoco
ccus hemolyticus) is a typical one. On the other hand, the history of mycoplasma research is relatively new, and in recent years, taxonomic, ecological or pathological studies have progressed, and the relationship between animals and mycoplasma has been gradually elucidated.
Mycoplasma are generally highly host specific and rarely infect across species. Furthermore, many mycoplasmas have relatively weak pathogenicity, are often isolated from healthy animals as normal flora, and their toxicity is considered to be relatively weak. It has also been reported that it acts mitogenically on lymphocytes (Y. Mizushima et a
I, Infect. Immun. Vol. tg, 636-645, 1985) It is expected to affect the immune function of the host.

Interferon (IFN) is a physiologically active protein produced by various cells such as lymphocytes and fibroblasts, and exhibits antiviral action and anticancer action. There are α, β, and γ subtypes of IFN, and α is at least 14 due to a slight difference in amino acid sequence and accompanying difference in antiviral action.
Classified into more than species. These IFNs are industrially produced by recent gene recombination technology and mass cell culture technology,
It is being clinically applied as an antiviral agent and an anticancer agent. In addition to these natural IFNs, novel artificial IFNs have also been produced by partially substituting the amino acid sequences of the above IFNs or combining the sequences of each IFN by gene recombination techniques.

Problems to be Solved by the Invention Although it has been well known that mycoplasma has an immunostimulating action as described above, no finding has been obtained that it is effective for cancer treatment.

In addition, various attempts have been made to combine IFN with other anticancer agents such as TNF, IL2, dacarbazine, ACNC (trade name Nidoran), etc., but there are no findings regarding the combination with treated Mycoplasma cells. , New and IFN
It provides a dosage form that is much more effective in treating tumors than when administered alone.

Means for Solving the Problems The present invention provides an antitumor agent containing a treated product of Mycoplasma cells as an active ingredient, and an antitumor agent containing IFN.

The species of mycoplasma that can be used in the present invention include gallisepticum and hyorhinis.
is, orale, fermentance
ans), hominis, mycoide
s), Pharringis (paryngis), Pneumoniae (pneu)
moniae), salivarium, etc., but not limited to these, and any mycoplasmas having immunostimulatory activity can be used, and these species may be used in combination.

The mycoplasma as described above can be cultured according to a conventional method. That is, commonly used medium such as PPLO medium, Frey's medium, Hayflick medium, Hank's solution, Chanock
Such as liquid medium and heart infusion medium,
Liquid medium containing serum, yeast extract, etc. of cattle, horses, pigs, rabbits, etc. as usual, about 30-39 ° C, preferably 37 ° C.
Static culture for 2 to 10 days. In some cases, it may be preferable to put a rubber stopper on the incubator or to perform the culture in a 5 to 10% carbon dioxide gas atmosphere. The cultured cells are separated by centrifugation or the like, washed with physiological saline or the like, and sterilized. The obtained mycoplasma cell-treated product may be concentrated or lyophilized, if desired. The treated cells are
It is desirable to keep it frozen.

Alternatively, cells infected with mycoplasma may be cultured and mycoplasma may be prepared from the culture supernatant.

The obtained treated product of mycoplasma cells has a remarkable anti-cancer effect even when it is administered alone, but when it is further administered with IFN, it exhibits a more excellent anti-cancer effect. The IFN that can be used in the present invention is not limited to a specific IFN, and it may be natural or artificial such as IFNα and its subtypes, IFNβ and IFNγ.
All IFNs can be used. However, if the IFN is species-specific, it should be used according to the subject of administration. Various interferons are already commercially available (for example, from LEE Biomolecular Research Laboratories, Inc.), and these commercially available products can be used. These IFNs can be used alone or in combination.

The obtained treated product of mycoplasma cells may be diluted or dissolved with physiological saline, which is generally used for injections, distilled water for injection, etc., and then administered to humans and animals intravenously, intramuscularly, intraperitoneally or subcutaneously. When administered to humans, symptoms, age,
Although it depends on the sex and the like, it is usually considered that the administration of about 10 ⁸ to 10 ¹¹ cells / Kg / day is effective. In addition, IFN
Further anti-cancer effect can be expected if the drug is added to the drug and administered at about 10 to 10 million units / day. The injection may contain a pharmaceutically acceptable preservative, stabilizer, preservative, and optionally a local anesthetic such as procaine.

Effect of the Invention The antitumor agent of the present invention comprises mycoplasma and IFN, and an immunostimulatory effect of mycoplasma on macrophages and the like, and a synergistic effect with the antitumor effect of IFN,
It has an excellent life prolonging effect and a cancer cell growth suppressing effect.

Examples Example 1 Preparation of Cancer Cells (MethA-R1 Strain) MethA-R1 cells are methylcholanthrene-induced fibrosarcoma derived from Balb / c mice, and were used by Uno et al. As a resistant strain to mouse interferons α, β and γ. It is a strain (K. Uno et al, 1985, Cancer Res. 45: 1320-1).
327).

2 × 10 ⁶ MethA-R1 cells were intraperitoneally transplanted into Balb / c mice, and 10 days later, they were collected from the peritoneal cavity. 10 syringes to collect
It was performed by injecting ml of Hank's solution (Hanks, Nissui) into the abdominal cavity of the mouse and recollecting the fluid accumulated in the abdominal cavity with a syringe. Collect the collected solution in a 50 ml centrifuge tube (Falc
on), centrifuge at 1000 rpm for 10 minutes, discard the supernatant,
It was suspended again in Hanks' solution. This cell suspension was used for the experiment.

Adjustment of Mycoplasma (Part 1) Mycoplasma gallicepticum
septicum, S-6 strain; hereinafter abbreviated as MG) was cultured and adjusted by the following method. Chicken serum PPLO medium (manufactured by Eiken: 100 g of bovine heart exudate, 10 g of peptone, 5 g of sodium chloride, 1 g of glucose, 0.25 g of thallium acetate dissolved in 1 L of water and adjusted to pH 7.8) with horse serum (sterilized by filtration to MG) Bacterial fluid (50 ml, number of bacteria approx. 10 ⁸ CFU / ml, CFU =), which was cultivated at 37 ° C for 2 days, in a medium (hereinafter referred to as horse serum supplemented medium) supplemented with 12% Colony Forming Unit) was used as the original culture solution. This bacterial solution was further added to approximately 2 L of horse serum-containing medium (3 L
(A triangular flask was used), and static culture was carried out at 37 ° C for 7 days. This culture solution was centrifuged at 8,000 rpm for 30 minutes, the supernatant was discarded, and the precipitated bacterial cells were suspended in physiological saline, followed by centrifugation at 16000 rpm for 20 minutes. This operation was repeated 3 times to wash the cells, and finally about 18 ml (2 × 10 ¹⁰ cells / ml) of a 100 times concentrated bacterial solution was obtained. The bacterial solution is sterilized at 120 ° C for 20 minutes and stored at -20 ° C.

Preparation of Mycoplasma (Part 2) The bacterial solution prepared by the above method and concentrated 100 times was lyophilized by the following procedure. That is, the MG bacterial solution was put in a glass bottle (diameter 3c
After filling 3 ml in m, height 6 cm), freeze-drying (vacuum freeze-dryer, Freezvac-4c-100S type, manufactured by Tozai Tsusho Co., Ltd.) was performed. After pre-freezing the sample bottle containing MG bacteria with dry ice / methanol (-50 ° C, about 20 minutes), attach it to the freeze dryer and operate it for about 6 hours at a vacuum degree of 10 ^-2 torr. After confirming that the sample became powdery, freeze drying was completed. 10 ⁹ per 0.2 mg of dried sample
It contained one MG strain.

Preparation of interferon IFNα (containing 1.8 million units) having mouse specificity was dissolved in physiological saline to make 9 ml, and an IFNα solution (200,000 units / ml) was prepared.

Mouse recombinant interferon γ (hereinafter, IFNγ
Is a ConA-induced T cell producing strain B5 strain (Y. Watanabe et al, Character) established by Watanabe et al.
ization of mouse IFNγ form a cloned T cell line, i
n the Biology of the Interferon System, 1983 (eds.
E.DcMaeyer and H.Schellekens), p.143-). 1 ml of the original preparation contains 1 mg IFNγ, 100 μg mouse serum albumin. This antiviral activity is 1 × 10 ⁷ units. An IFNγ solution (100,000 units / ml) was prepared by dissolving 0.1 ml of the IFNγ preparation in physiological saline to make 10 ml.

Experimental method and results MethA-R1 cells collected by the above method were 4 × 10 ⁶ cells / ml.
Diluted with a 2.5 ml syringe with a 23G needle
Balb / c mice are intraperitoneally implanted with 2 × 10 ⁶ mice per mouse. 10 ^{9 of the} above-mentioned MG bacterium (Adjustment 1) or its freeze-dried product (Adjustment 2), or 20,000 units of IFN
γ or 50,000 units of IFNα is administered alone, or MG bacteria and interferon are mixed and administered.

-Preparation of MG solution (1) Dilute 2 ml of the bacterial solution (2 x 10 ¹⁰ cells / ml) obtained in Preparation 1 with physiological saline to make 10 ml (4 x 10 ⁹ cells / ml). The MG solution was mixed with an equal volume of physiological saline (1 × 10 ⁹ pieces / 0.5 m
l).

・ Preparation of MG solution (Part 2) 8 m of freeze-dried product of bacteria (5 × 10 ⁹ cells / mg) prepared in Preparation part 2
Dilute g with physiological saline to make 10 ml (4 × 10 ⁹ cells / ml). The MG solution was mixed with an equal volume of saline (1 x 10 ⁹ /
0.5 ml).

・ Preparation of MG solution containing IFN IFNα solution (200,000 units / ml) obtained by the above preparation of IFN
Or IFNγ solution (100,000 units / ml) is mixed with an equal volume of the above MG solution (4 × 10 ⁹ pieces / ml), and IFNα (50,000 units / 0.5 ml)
Or IFNγ (25,000 units / 0.5ml) and MG (1 × 10 ⁹ pieces / 0.5m)
The solution containing l) was prepared.

・ Preparation of IFN solution IFNα solution (200,000 units / ml) obtained by the above-mentioned preparation of IFN
Alternatively, an IFNγ solution (100,000 units / ml) was mixed with an equal amount of physiological saline to prepare a solution containing IFNα (50,000 units / 0.5 ml) or IFNγ (25,000 units / 0.5 ml).

Administration was carried out every 3 days after 3 days, with the above MG and / or IF.
A solution in which Nγ or IFNα was suspended was injected into the abdominal cavity of mice using 0.5 ml per mouse and a 2.5 ml syringe with a 23G needle.

Balb / c mice were 6 to 15 in each group. After the 10th administration of MG bacterium or interferon was completed, the animals were kept in the same state and the survival condition was observed.

The average survival time was calculated along with the survival rate until the end of the test (35 days). The average number of days to live is a value obtained by calculating the arithmetic mean value and the deviation value of the reciprocal of the number of days to live of each individual, and then calculating the reciprocal thereof. The results are shown in Table 1.

A sufficient effect was not obtained by the administration of 50,000 units of IFNα, but a significant life-prolonging effect was observed with the administration of ⁹ MG10 ⁹ units. By simultaneous administration of 10 ⁹ MG bacteria and 50,000 units of IFNα,
An even more significant life-prolonging effect was observed as compared to the single administration of. Also, IFNγ administration of 25,000 units has a life-prolonging effect to some extent, and a combination of 10 ⁹ MG's can cure all cases.
An excellent antitumor effect was obtained.

From the above results, even if MG alone is administered, there is a life-prolonging effect, and IFN
It was proved that the combination with α or IFNγ showed a more significant antitumor effect.

Example 2 Cancer cells were prepared in the same manner as in Example 1. Mycoplasma was prepared by the adjustment (No. 1) described in Example 1.
In addition to the bacteria, those produced by the method described below were used.

Adjustment of Mycoplasma (3) Mycoplasma hyorhin
About 10% of is, BTS-7 strain, abbreviated as MH hereinafter to BHL medium (composition described below) was used (passage of the preserved bacterial solution used in BHL medium was preserved at -70 ° C) Cell culture solution, after culturing at 37 ° C for 3 days, further diluting the culture medium twice and culturing
20 ml was obtained. 2000 ml of BHL medium (3 L volume of 3
(A square flask was used), a rubber stopper was added, and the mixture was incubated at 37 ° C. with stirring with a magnetic stirrer. After 3 days of culturing, the cells were collected by centrifuging at 8000 rpm for 30 minutes while cooling and sterilized with PBS.
(NaH ₂ PO ₄・ H ₂ O 0.23g, Na ₂ HPO ₄・ 12H ₂ O 1.19g, NaCl
Re-float with 8.5 g, distilled water 1000 ml, pH 6.8) 2 at 15000 rpm
Centrifugation was performed for 0 minutes. This operation was repeated 3 times for washing, then, steam sterilization treatment was carried out at 120 ° C for 20 minutes and storage at -20 ° C. The concentration of the bacterial solution is 2 × 10 ¹⁰ cells / ml.

BHL medium BHL basal medium ^a) 750 ml pig serum ^b) 100 ml horse serum ^b) 100 ml 25% yeast extract ^c) 50 ml 5% sodium carbonate pH 7.8 corrected a) BHL basal medium (115 ℃, sterilized by autoclave for 15 minutes) ) Brucella broth (Gibco) 11.6g Lactalbumin hydrolyzate 4.0g Salt solution (× 10) ^# 100 ml Distilled water 1400 ml b) Serum (filter sterilized) Porcine serum (Flow Lab.) Horse serum (Osaka Univ.) (Ciken) c) Yeast extract liquid Dry yeast (Nicken) 500g Distilled water 1500ml After heating in boiling water for 20 minutes, centrifuge at 7,000-8000 rpm for 20 minutes, and the supernatant was adjusted to pH 7.6 with 4% sodium hydroxide and filtered. sterilization is # a 1000ml with saline (× 10) NaCl 80.0g KCl 4.0g Na 2 HPO 4 · 12H 2 O 1.2g KH 2 PO 4 0.6g glucose 20.0 g 0.5% phenol red 40.0ml distilled water, 1
Sterilization by autoclave for 10 minutes at 15 ℃ Preparation of Mycoplasma (Part 4) MH or Mycoplasma olere
leth) -infected MethA-R1 cells 2.5 × 10 ⁵ cells were added to 10 ml of culture medium [RPMI-1640 (Nissui), 10% fetal bovine serum (Hyklorele) was added, and sodium bicarbonate was added to 1 L per 1 L.
3g, 1.2g of HEPES was added], and put in a plastic Petri dish (10cm in diameter) to prepare 20 pieces. Three
After culturing for a day, the culture solution was collected together with the cells, and the culture was performed at 1000 rpm for
Centrifuge for minutes to remove cells. Further, add the supernatant to 30000rp
Ultracentrifuge for 30 minutes at m. Mycoplasma-derived components are contained in the supernatant, and mycoplasma cells are contained in the sediment. The precipitate was further washed with physiological saline three times, suspended, counted, sterilized by steam at 120 ° C for 10 minutes, and stored at -70 ° C. The bacterial solution contains 10 ¹⁰ cells / ml.

Experimental method and results 23G of MethA-R1 cells diluted to 4 × 10 ⁶ cells / ml
2x per mouse in the abdominal cavity of Balb / c mice with a syringe with needle
Transplant 10 ⁶ . Every 3 days after every 2 days, MG [adjustment (1)] 2 x 10 ⁸ pieces or MH [adjustment (3) or (4)] 2 x 10 ⁸ pieces in 0.5 g with a syringe with a 23G needle ml
Each was intraperitoneally administered, and 8 days later, the total number of MethA-R1 cells in the abdominal cavity was counted. The MG or MH was adjusted by diluting with physiological saline as in Example 1.

Counting MethA-R1 cells After injecting 10 ml of Hank's solution using a syringe with a 20G needle into the abdominal cavity of a mouse exsanguinated from the tibial artery, collect the solution as much as possible using the same syringe again, and use the The number of cancer cells per 1 ml was counted using a hemocytometer, and the total number of cells in mouse abdominal cavity in 10 ml of Hanks' solution was calculated. When ascites accumulated and the collected liquid exceeded 9 ml, 1 ml was added to the collected amount, and the number of cancer cells per ml was added to obtain the total cell number. The results are shown in Table 2. As of the 8th day
Regardless of the type of MG, MH, or the adjustment method of MH,
Administration of mycoplasma significantly suppressed the growth of MethA-R1 cells.

Example 3 The method for adjusting cancer cells (MethA-R1) and IFNα is the same as in Example 1. As the mycoplasma, MH and M. orale prepared by the preparation method (4) of the mycoplasma shown in Example 2 were used.

Experimental Method and Results MethA-R1 cells were diluted and suspended in Hanks' solution at 4 × 10 ⁷ cells / ml, and 25 μl (10 ⁶ cells) each was put into the hind footpad of Balb / c mice using 100 μl microsyringe. Give an injection. Three
MH2 × 10 on the foot pads transplanted with cancer cells every other day
^Eight , M. orale 2 × 10 ⁸ and 10,000 units of IFNα were used alone or in combination in a 1 ml syringe with a 27G cloak needle. The injection volume is 50 μl. Swelling of the footpad was indicated by the thickness of the footpad measured with a caliper (dial caliper). Measurements were made every other day before injection. The results are shown in FIG. The data shown here are the average values of the footpad thickness of 6 animals. The size of the solid cancer due to the actual proliferation of MethA-R1 cells is comparable to the value of the third power of the footpad thickness.

MH and M. orale were also effective alone in solid cancer. Some effects were observed even when 10,000 units of IFNα were administered alone. Administration of IFNα in combination with MH or M. orale markedly suppressed the growth of cancer cells.

Safety with Mycoplasma gallisepticum (MG) in combination with IFNα Safety studies were conducted using the tail vein inoculation method in mice. The test method was 4 weeks old dds mice (female, body weight 18-2
Into the tail vein of 0 g), 0.4 ml per animal was inoculated with a mixture of MG and IFNα in the following mixing ratio (5 animals per group were used).
The syringe used for inoculation was a 1 ml syringe with a luer needle 1/5 vein needle set. The mixing ratio of MG and IFNα is as follows.

1.MG 0.2mg ＋ IFNα 50,000 units / 0.4ml / mouse 2.MG 2mg ＋ IFNα 500,000 units / 0.4ml / mouse 3.MG 10mg ＋ IFNα 2.5 million units / 0.4ml / mouse 4. Sterile saline 0.4ml / mouse , Freeze-drying (adjustment of mycoplasma (2),
Samples (containing 10 ⁹ cells / 0.2 mg) were dissolved in sterilized physiological saline and adjusted to a required concentration for use.

Results: In each group, mice dying until the 12th day after the inoculation and side effects (anorexia, piloerection, etc.) were not observed.

EFFECTS OF THE INVENTION The antitumor agent of the present invention shows a remarkable life-prolonging effect and cancer cell inhibitory effect in experiments using mice, and is an effective antitumor agent for humans and animals.

[Brief description of drawings]

FIG. 1 shows changes in footpad thickness when IFN and / or mycoplasma cells-treated products were administered to mouse footpads transplanted with MethA-R1 cells.

Claims (4)

[Claims] 1. An antitumor agent comprising a treated product of mycoplasma cells as an active ingredient. 2. The antitumor agent according to claim 1, wherein the mycoplasma is Mycoplasma gallisepticum, Mycoplasma hyorhinis and / or Mycoplasma olere. 3. The antitumor agent according to claim 1, further comprising interferon as an active ingredient. 4. The interferon is interferon α
And / or interferon γ, The antitumor agent according to claim 3.