GB2072502A - Antitumour agent comprising lactobacillus casei - Google Patents

Abstract

Immunostimulating agents for inhibiting tumours comprise Lactobacillus casei YIT 9018.

Description

SPECIFICATION Antitumour agent This invention relates to an antitumour agent.

The invention provides an antitumour agent containing the strain Lactobacillus caseiYIT 9018 (Deposit No. FERM-P 4751) as an effective ingredient. This strain is deposited at the Fermentation Research Institute, Government Industrial Research, Ministry of International Trade and Industry, Japan and is available to the public.

While the antitumour activity of an alive streptococcal preparation (Japan Tokkyo Koho, 6690, 1968) or the extract from lactobacilli (Japan Tokkyo Koho, 28558, 1970) has been reported, it has not previously been known that the heat-killed whole cell preparation of the strain YIT 901 8 has an antitumour activity.

This invention is concerned not only with the antitumour activity of alive but also heat-killed Lactobacillus caseiYIT 9018 whole cells. This provides the following benefits: the process of the preparation is simpler and the possible side effects due to the presence of additives for maintaining the survival of the bacteria can be prevented. It also deserves a special emphasis that Lactobacillus caseiYIT 9018 is non-toxic and used in practice in a fermented milk product.

More specifically, it has been found that Lactobacillus casei YIT 901 8 (Deposit No.

FERM-P4751) (hereinafter referred to as "LC 9018") has a higher antitumour activity compared to other lactobacilli. The bacterial strain exhibits antitumour activity against solid and ascites tumour of Sarcoma 1 80 upon intravenous, subcutaneous, oral or intraperitoneal administration in mice. Furthermore, it shows an antitumour activity against mouse leukemia L1 210 or P-388 on which commercial antitumour streptococcal preparations have no effect. In addition, the toxicity of LC 9018 is significantly lower than those of other lactobacilli and streptococcal antitumour agents.

LC 9018 has the following characteristics: Cell shape short rod Gram's stain positive Optimal pH for growth 6.6-7.0 (at 35-39"C) Range of temperature for growth 15-42"C Methyl red test negative Voges-Proskauer reaction negative Production of indole negative Production of H2S 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 from glucose negative Fermentation arabinose negative xylose negative rhamnose negative glucose positive man nose positive lactose 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 ordinal methods. For example, it can be cultivated on a semisynthetic broth containing lactose, glucose peptone, yeast extract, KH2PO4, K2HPO4, MgSO4 etc. The culvitation is suitably performed at 37"C for 18 to 24 hours. After cultivation, bacterial cells can be collected by centrifugation and washed with distilled water and lyophilized. While the bacterial cells require no additives for survival, drugs or other substances might be added for maintaining quality, if desired.

LC 9018 is preferably cultivated using the broth shown in Table A. The amounts are per 1 000 ml of distilled water.

Table A Trypticase 10 9 Yeast extract 5 g Tryptose 3 9 K2HPO4 3 9 KH2PO4 3 g Ammonium citrate 2 g Sodium acetate 1 g Tween 80 80 g Glucose 20 g Cystein 0.2 g MgSO4-7H20 0.5 g FeSO4-7H20 O.04g MnSO4-2H20 0.12 g After cultivation, at 37"C for 20 to 40 hours, the cells can be washed with distilled water and lyophilized.

LC 901 8 is preferably suspended in physiological saline for use as an injection. The daily effective doses are normally 30 to 100 mg/kg (ideally 50 mg/kg) for intravenous injection, 5 to 20 mg/kg (ideally 10 mg/kg) for intraperitoneal injection and 1 000 to 2000 mg/kg for oral administration. The frequency and duration of the administration should be varied according to the condition of a patient.

As mentioned above, not only live but also heat-killed LC 9018 has a significant antitumour activity and is not pathogenic. The use of 9018 may bring less restrictions on the preparative process and its clinical usage and preparation can be effected econimically.

The present invention is illustrated by the following Experiments and Examples.

EXPERIMENT 1: ANTITUMOUR ACTIVITY OF LC9018 Example (1) Sarcoma 180 (1-2 X 106 cells/mouse) was implanted into ICR male mice subcutaneously on day 0. The 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 tumor was measured. The inhibition rate was culculated according to a formula: Average tumor weight of tested mice Inhibition rate = (1 - ) X 100.

Average tumor weight of control mice As shown in Table 1, lactobacilli had the inhibition rates of 40-70% as well as the streptococcal preparation. LC 9018 showed the inhibition rate of more than 80%.

Example (2) Sarcoma 180(1-2 x 106 cells/mouse) was implanted subcutaneously into male ICR mice after admixed with 1 mg of LC 9018. The inhibition rate with LC 9018 was 97.3% which determined as described in Example (1).

Example (3) Sarcoma 1 80 (1-2 x 106 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 901 8 significantly inhibited the growth of Sarcoma 180 at the dose of 40 mg/kg.

Example (4) Sarcoma 1 80 (1-2 X 106 cells/mouse) was inoculated subcutaneously into male ICR mice on 'day 0. LC 9018 was given orally daily on days - 10, + land + 10. It was found LC 9018 showed antitumor activity even by oral administration (Table 3).

Example (5) Sarcoma 180 (1-2 X 106 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: Average survival time of tested mice T/C % = x 100.

Average survival time of control mice As shown in Table 4, LC 9018 increased the survival time of mice implanted tumor cells.

Example (6) Mouse leukemia L1210 or Put 388 (1 x 105 cells/mouse) was inoculated intraperitoneally into male BDF, 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 LC9018 (1) LD50 The LD50 of lactobacilli which showed the inhibition rate (Table I) of more than 40% was determined using ICR mice according to the method of Lithfield-Wilcoxon. LC 9018 was less toxic compared to streptococcal preparation as well as other lactobacilli.

(2) Antigenicity LC 901 8 was injected subcutaneously into white guinea pig for three times every 3 days at the total does 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 901 8 showed negative reactions in both tests.

EXPERIMENT 3: ANTITUMOUR ACTIVITY AND TOXICITY OF HEAT-KILLED CELLS LC 9018 The antitumour activity and the toxicity of heat-killed LC 9018 were same as those alive cells.

EXAMPLE OF PREPARATION (1) LC 9018 (1 x 107 cells) was inoculated into 1000 ml of the broth of which composition was mentioned above and cultivated at 37"C for 20 hr. The final alive cell number reached at maximum (2.5 X 109/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 leaved at 5"C for 30 days, LC 901 8 in ampoules was used for the antitumour experiment.

The inhibition rate was 83.2%.

(2) LC 9018 was obtained as in (1). It was autoclaved at 121"C for 20 min and dryed at 80"C. The powder was divided into ampoules and stored at 5"C for 2 months. The inhibition rate against Sarcoma 180 with this preparation was 78.5% and the LD50 was 620 mg/kg or 720 mg/kg in male or female ICR mice, respectively.

(3) The culture broth of 300 ml containing 2.5 x 109 cells/ml of LC 9018 was inoculated into 10 1 of Rogosa's medium. The cell number was 2.3 X 109/ml after the cultivation at 37"C for 20 hr. The wet cells (165 g) were dryed at 80"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 dryness. 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 Sarcoma 180 (1-2 X 106 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 lactobacilli Strain or preparation Inhibition rate* L. casei YIT 9018 (LC 9018) 82.7 N N Vl11-1 65.5 TK to IV-2 66.6 L. acidophilus B-3208 23.0 ,, YIT 0163 43.5 L. salivarius YIT 0089 21.5 ,, YIT 0104 40.9 L. fermentum YIT 0082 14.9 ,, YIT 0159 10.2 L. plantarum YIT 0102 17.2 ,, YIT 0158 40.4 L. bulgaricus YIT 0046 64.7 L. jugurti YIT 0085 17.7 L. helveticus YIT 0083 29.0 L. lactis YIT 0086 1 2.8 L. leichmannii YIT 0087 28.4 L. delbrueckii YIT 0080 47.9 L. brevis YIT 0076 5.3 L. jensenii YIT 0084 38.4 Streptococcal preparation 50.8 Sarcoma 180 (1-2 x 106 cells/mouse) was implanted into ICR mice 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.

Average tumor weight of tested mice *Inhibition rate = (1 - ) x 100.

Average tumor weight of control mice Table 2. Antitumor activity of subcutaneously administered LC 9018 Dose (mg/kg) Inhibition rate* O (Control) O 4 39.3 40 84.4 Sarcoma 180 (1-2 x 106 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 3 weeks after the inoculation and the weight of tumor was measured.

*Inhibition rate was calculated as Table 1.

Table 3. Antitumor activity of orally administered LC 9018 Total dose (mg/kg) Inhibition rate* O (Control) O 1200 70.5 2000 64.2 Sarcoma 1 80 (1-2 x 105 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 tomur was measured.

'Inhibition rate was calculated as Table 1.

Table 4. Antitumor activity of intraperitoneally administered LC 9018 against intraperitoneally inoculated Sarcoma 180 Total dose (mg/kg) T/C %* O (Control) 100 1 104 10 367 40 243 Sarcoma 180 (1-2 X 106 cells/mouse) was inoculated intraperitoneally on day 0. LC 9018, suspended in saline, was injected intraperitoneally on daily on days - 5- - 1.

Average survival time of tested mice *T/C % = X 100.

Average survival time of control mice Table 5. Antitumor activity of LC 9018 against mouse leukemia Leukemia 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 P-388 (1 X 105 cells/mouse) was inoculated intraperitoneally into BDF, mice on day 0. LC 9018, suspended in saline, was given intraperitoneally daily on days + 1- + 5.

*T/C% was calculated as Table 4.

**Pc O.005, ***P < 0.0001.

Table 6. Acute toxicity of lactobacilli and streptococcal preparation LDso (mg/kg) Route Strain or preparation male female i.p.

L. casei YIT 9018 (LC 9018) 650 730 ,, N Vlil-1 515 581 ,, TK IV-2 350 466 L. acidophilus YIT 0163 550 612 L. salivarius YIT 0153 504 566 L. plantarum YIT 0158 340 361 L. bulgaricus YIT 0046 310 358 L. delbrueckii YIT 0080 256 283 Streptococcal preparation 1 25 140 i.v.

L. casei YIT 9018 (LC 9018) 156 240 Streptococcal preparation 24.5 25.5 s.c.

L. casei YIT 9018 (LC 9018) 2500 2500 Streptococcal preparation 238 1 97 p.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 9) intraperitoneally (i.p.), intravenously (i.v.), subcutaneously (s.c.) or orally (p.o.).

Claims (1)

1. Lactobacillus casei YIT 901 8, for use in a method of treatment of the human or animal body by surgery or therapy.