Antitumor agent containing Lactobacillus casei YIT 9018

Abstract

Immunostimulating agent for inhibiting tumors comprising Lactobacillus casei YIT 9018 and methods for treating and/or preventing tumor growth.

Description

BACKGROUND OF THE INVENTION

This invention relates to an antitumor agent containing Lactobacillus casei YIT 9018 (Deposit No. FERM-P 4751) as an effective ingredient. This strain is deposited at Fermentation Research Institute, Government Industrial Research, Ministry of International Trade and Industry, Japan.

While the antitumor activity of a live streptococcal preparation (Japan Tokkyo Koho, 6690, 1968) or an extract from lactobacilli (Japan Tokkyo Koho, 28558, 1970) has been reported, it has not been known that the heat-killed whole cell preparation has an antitumor activity.

SUMMARY OF THE INVENTION

This invention is concerned with the antitumor activity of not only live but also heat-killed Lactobacillus casei YIT 9018 whole cells. This provides the following benefits: the process of preparation is simpler, and the side effects of the additives for maintaining the survival of the bacteria can be prevented. It also deserves a special emphasis that Lactobacillus casei YIT 9018 is non-toxic and is practically used in a fermented milk product.

DETAILED DESCRIPTION OF THE INVENTION AND ITS PREFERRED EMBODIMENTS

It has been shown that Lactobacillus casei YIT 9018 (Deposit No. FERM-P 4751) (hereinafter referred to as "LC 9018") has a higher antitumor activity compared to other lactobacilli. This bacterial strain exhibited antitumor activity against solid and ascites tumor of Sarcoma 180 with intravenous, subcutaneous, oral or intraperitoneal administration in mice. Furthermore, it showed an antitumor activity against mouse leukemia L1210 or P-388 on which commercial antitumor streptococcal preparation had no effect. In addition, the toxicity of LC 9018 was significantly lower than those of other lactobacilli and streptococcal antitumor agents.

LC 9018 was cultivated on a broth containing (per 1000 ml of distilled water)

Trypticase--10 g Yeast extract--5 g Tryptose--3 g K.sub.2 HPO.sub.4 --3 g KH.sub.2 PO.sub.4 --3 g Ammonium citrate--2 g Sodium acetate--1 g Tween 80 --80 g Glucose--20 g Cystein--0.2 g MgSO.sub.4 -7H.sub.2 O--0.5 g FeSO.sub.4 -7H.sub.2 O--0.04 g MnSO.sub.4 -2H.sub.2 O--0.12 g at 37.degree. C. for 20-40 hr. After cultivation, the cells were washed with distilled water and lyophilized.

EXPERIMENT 1: ANTITUMOR ACTIVITY OF LC 9018

EXAMPLE (1)

Sarcoma 180 (1-2.times.10.sup.6 cells/mouse) was implanted into ICR male mice subcutaneously on day 0. A suspension of lactobacilli in saline (0.25 mg/mouse) was injected intravenously daily on days +1-+5. Mice were dissected 3 weeks after the tumor implantation and the weight of the tumor was measured. The inhibition rate was calculated according to the formula: ##EQU1## As shown in Table 1, other lactobacilli had inhibition rates of 40-70% as did the streptococcal preparation. LC 9018 showed an inhibition rate of more than 80%.

EXAMPLE (2)

Sarcoma 180 (1-2.times.10.sup.6 cells/mouse) was implanted subcutaneously into male ICR mice after being admixed with 1 mg of LC 9018. The inhibition rate with LC 9018 was 97.3%, determined as described in Example (1).

EXAMPLE (3)

Sarcoma 180 (1-2.times.10.sup.6 cells/mouse) was inoculated subcutaneously followed by the subcutaneous injection of LC 9018 24 hr after the tumor implantation. As shown in Table 2, LC 9018 significantly inhibited the growth of Sarcoma 180 at the dose of 40 mg/kg.

EXAMPLE (4)

Sarcoma 180 (1-2.times.10.sup.6 cells/mouse) was inoculated subcutaneously into male ICR mice on day 0. LC 9018 was given orally daily on days -10, +1, and +10. It was found LC 9018 showed antitumor activity even by oral administration (Table 3).

EXAMPLE (5)

Sarcoma 180 (1-2.times.10.sup.6 cells/mouse) was inoculated intraperitoneally on day 0. LC 9018, suspended in saline, was injected intraperitoneally daily on days -5--1. The relative survival rate (T/C %) was calculated as follows: ##EQU2## As shown in Table 4, LC 9018 increased the survival time of mice with implanted tumor cells.

EXAMPLE (6)

Mouse leukemia L1210 or P-388 (1.times.10.sup.5 cells/mouse) was inoculated intraperitoneally into male BDF.sub.1 mice on day 0. LC 9018, suspended in saline, was given intraperitoneally daily on days +1-+5. As shown in Table 5, LC 9018 also inhibited the growth of leukemia cells. This should be emphasized because the commercial streptococcal preparation has no antitumor activity against leukemia.

EXPERIMENT 2: TOXICITY OF LC 9018

(1) LD.sub.50

The LD.sub.50 of lactobacilli which showed an inhibition rate (Table 1) of more than 40% was determined using ICR mice according to the method of Litchifield-Wilcoxon. LC 9018 was less toxic compared to streptococcal preparation as well as other lactobacilli (Table 6).

(2) Antigenicity

LC 9018 was injected subcutaneously into white guinea pigs three times every 3 days at the total dose of 50 mg/kg. The anaphylactic reaction test and agglutination test were carried out 10 days and 12 days after the final injection of LC 9018, respectively. All the guinea pigs given LC 9018 showed negative reactions in both tests.

EXPERIMENT 3: ANTITUMOR ACTIVITY AND TOXICITY OF HEAT-KILLED LC 9018

The antitumor activity and the toxicity of heat-killed LC 9018 were the same as those of live cells.

LC 9018 has the following characteristics:

Cell shape--short rod Gram's stain--positive Optimal pH for growth--6.6-7.0 (at 35.degree.-39.degree. C.) Range of temperature for growth--15.degree.-42.degree. C. Methyl red test--negative Voges-Proskauer reaction--negative Production of indole--negative Production of H.sub.2 S--negative Ammonia from arginine--negative Reduction of nitrate--negative Production of catalase--negative Liquefaction of gelatin and casein--negative Citrate utilization--negative Coagulation of milk--positive Reduction of litmus milk--positive Utilization of ammonium and urea--negative Gas frpm glucose--negative Fermentation arabinose--negative xylose--negative rhamnose--negative glucose--positive mannose--positive galactose--positive sucrose--positive maltose--positive lactose--positive cellobiose--positive trehalose--positive melibiose--negative raffinose--negative melezitose--positive mannitol--positive sorbitol--positive salicin--positive amygdalin--positive LC 9018 can be cultured according to an ordinary method. For example, it can be cultivated on a semisynthetic broth containing lactose, glucose peptone, yeast extract, KH.sub.2 PO.sub.4, K.sub.2 HPO.sub.4, MgSO.sub.4 etc. The cultivation was performed at 37.degree. C. for 18-24 hr. After cultivation, bacterial cells were collected by centrifugation and washed with distilled water and lyophilized. While the bacterial cells required no additives for maintaining their survival, it is possible to add certain drugs or substances for maintaining quality as needed.

LC 9018 was suspended in physiological saline for use as an injection. The daily effective doses were 30-100 mg/kg (ideally 50 mg/kg) for intravenous injection, 5-20 mg/kg (ideally 10 mg/kg) for intraperitoneal injection and 1000-2000 mg/kg for oral administration. The frequency and duration of the administration should be varied according to the condition of the patient.

As mentioned above, not only live but also heat-killed LC 9018 had a significant antitumor activity, and it was not pathogenic. Also, the process for preparing, and the clinical usages of, LC 9018 preparations, are not limited, and could be accomplished economically.

EXAMPLE OF PREPARATION

(1) LC 9018 (1.times.10.sup.7 cells) was inoculated into 1000 ml of the broth mentioned above and was cultivated at 37.degree. C. for 20 hr. The final live cell number reached at maximum (2.5.times.10.sup.9 /ml). The cells were collected by centrifugation and washed with distilled water. The suspension of LC 9018 in distilled water was divided in ampoules and lyophilized. After storing at 5.degree. C. for 30 days, LC 9018 in ampoules was used for the antitumor experiment. The inhibition rate was 83.2%.

(2) LC 9018 was obtained as in (1). It was autoclaved at 121.degree. C. for 20 min and dried at 80.degree. C. The powder was divided into ampoules and stored at 5.degree. C. for 2 months. The inhibition rate against Sarcoma 180 with this preparation was 78.5% and the LD.sub.50 was 620 mg/kg or 720 mg/kg in male or female ICR mice, respectively.

(3) The culture broth, 300 ml, containing 2.5.times.10.sup.9 cells/ml of LC 9018 was inoculated into 10 liters of Rogosa's medium. The cell number was 2.3.times.10.sup.9 /ml after the cultivation at 37.degree. C. for 20 hr. The wet cells (165 g) were dried at 80.degree. C. for 3 hr and 40 g of powder was obtained. The powder was added to 40 ml of hydroxypropyl cellulose solution (10%) in ethanol and granulated followed by drying. The granules were given orally at the dose of 0.3 g/kg/day for 10 days to ICR mice which had been implanted subcutaneously with Sarcoma 180 (1-2.times.10.sup.6 cells/mouse) 10 days before the oral administration of the granules. The inhibition rate at 3 weeks after the tumor implantation was 68.2%.

TABLE 1______________________________________Antitumor activity of lactobacilliStrain or preparation Inhibition rate*______________________________________L. casei YIT 9018 (LC 9018) 82.7L. casei N VIII - 1 65.5L. casei TK IV - 2 66.6L. acidophilus B-3208 23.0L. acidophilus YIT 0163 43.5L. salivarius YIT 0089 21.5L. salivarius YIT 0104 40.9L. fermentum YIT 0082 14.9L. fermentum YIT 0159 10.2L. plantarum YIT 0102 17.2L. plantarum YIT 0158 40.4L. bulgaricus YIT 0046 64.7L. jugurti YIT 0085 17.7L. helveticus YIT 0083 29.0L. lactis YIT 0086 12.8L. leichmannii YIT 0087 28.4L. delbrueckii YIT 0080 47.9L. brevis YIT 0076 5.3L. jensenii YIT 0084 38.4Streptococcal preparation 50.8______________________________________ Sarcoma 180 (1-2 .times. 10.sup.6 cells/mouse) was implanted into ICR mic subcutaneously on day 0. The suspension of lactobacilli or streptococcal preparation in saline (0.25 mg/mouse) was injected intravenously daily on days +1- +5. ##STR1## TABLE 2______________________________________Antitumor activity of subcutaneously adminis-tered LC 9018Dose (mg/kg) Inhibition rate*______________________________________0 (Control) 04 39.340 84.4______________________________________ Sarcoma 180 (1-2 .times. 10.sup.6 cells/mouse) was inoculated subcutaneously into ICR mice. LC 9018, suspended in saline, was injected subcutaneously 24 hr after the tumor inoculation. Animals were dissected weeks after the inoculation and the weight of the tumor was measured. *Inhibition rate was calculated as for Table 1. TABLE 3______________________________________Antitumor activity of orally administeredLC 9018Total dose (mg/kg) Inhibition rate*______________________________________0 (Control) 01200 70.52000 64.2______________________________________ Sarcoma 180 (1-2 .times. 10.sup.6 cells/mouse) was inoculated subcutaneously into ICR mice on day 0. LC 9018 was given orally daily on days -10, +1, and +10. Animals were dissected 3 weeks after the inoculation of tumor cells and the weight of the tumor was measured. *Inhibition rate was calculated as for Table 1. TABLE 4______________________________________Antitumor activity of intraperitoneallyadministered LC 9018 against intraperitoneallyinoculated Sarcoma 180Total dose (mg/kg) T/C %*______________________________________0 (Control) 1001 10410 36740 243______________________________________ Sarcoma 180 (1-2 .times. 10.sup.6 cells/mouse) was inoculated intraperitoneally on day 0. LC 9018, suspended in saline, was injected intraperitoneally daily on days -5--1. ##STR2## TABLE 5______________________________________Antitumor activity of LC 9018 against mouseleukemiaLeukemia cell Total dose (mg/kg) T/C %*______________________________________L1210 0 (Control) 100 6 119 12 128** 60 138*** 120 127**P-388 0 (Control) 100 5 120** 50 118** 250 108______________________________________ Mouse leukemia L1210 or P388 (1 .times. 10.sup.5 cells/mouse) was inoculated intraperitoneally into BDF.sub.1 mice on day 0. LC 9018, suspended in saline, was given intraperitoneally daily on days +1-+5. *T/C % was calculated as for Table 4. **P < 0.005, ***P < 0.0001. TABLE 6______________________________________Acute toxicity of lactobacilli and strepto-coccal preparation LD.sub.50 (mg/kg)Route Strain or preparation male female______________________________________i.p. L. casei YIT 9018 (LC 9018) 650 730 L. casei N VIII - 1 515 581 L. casei TK IV - 2 350 466 L. acidophilus YIT 0163 550 612 L. salivarius YIT 0104 504 566 L. plantarum YIT 0158 340 361 L. bulgaricus YIT 0046 310 358 L. delbrueckii YIT 0080 256 283 Streptococcal preparation 125 140i.v. L. casei YIT 9018 (LC 9018) 156 240 Streptococcal preparation 24.5 25.5s.c. L. casei YIT 9018 (LC 9018) 2500 2500 Streptococcal preparation 238 197p.o. L. casei YIT 9018 (LC 9018) 5000 5000 Streptococcal preparation 500 500______________________________________ Lactobacilli or streptococcal preparation was given to ICR mice (body weight of about 25 g) intraperitoneally (i.p.), intravenously (i.v.), subcutaneously (s.c.) or orally (p.o.).

Claims

1. A composition for inhibiting tumor growth, which comprises a tumor growth-inhibiting amount of whole cells of Lactobacillus casei YIT 9018 and a physiologically acceptable saline carrier.

2. A composition according to claim 1, wherein the Lactobacillus casei YIT 9018 is heat-killed Lactobacillus casei YIT 9018 whole cells.

3. A composition for inhibiting tumor growth, which comprises a tumor growth-inhibiting amount of whole cells of Lactobacillus casei YIT 9018 and hydroxypropyl cellulose.

4. A composition according to claim 3, wherein the Lactobacillus casei YIT 9018 is heat-killed Lactobacillus casei YIT 9018 whole cells.

5. A method of inhibiting tumor growth in an animal, which comprises administering to the animal a tumor growth-inhibiting amount of whole cells of Lactobacillus casei YIT 9018.

6. A method according to claim 5, wherein the Lactobacillus casei YIT 9018 is heat-killed Lactobacillus casei YIT 9018 whole cells.

7. A method according to claim 5 or 6, wherein the Lactobacillus casei YIT 9018 is administered in admixture with a physiologically acceptable saline carrier.

8. A method according to claim 5 or 6, wherein the Lactobacillus casei YIT 9018 is administered in admixture with hydroxypropyl cellulose.