Abstract:
A carcinostatic agent having an immunopotentiating carcinostatic effect which contains as an active ingredient Eisenin (tripeptide) of the general formula: 
     
       L-Pyroglu-L-Glu-L-Ala 
     
     wherein Pyroglu, Glu and Ala represent pyroglutamic acid, glutamic acid and alanine respectively.

Description:
BACKGROUND OF THE INVENTION 
     This invention relates to a carcinostatic agent having an immunopotentiating carcinostatic effect which contains as an active ingredient Eisenin which is a tripeptide obtained by extraction from seaweed Eisenia bicyclis or by synthesis, and more specifically, it relates to a carcinostatic agent having an immunopotentiating carcinostatic effect which contains as an active ingredient Eisenin (tripeptide) of the general formula: 
     
         L-Pyroglu-L-Gln-L-Ala 
    
     wherein Pyroglu, Gln and Ala represent pyroglutamic acid, glutamine and alanine, respectively. 
     SUMMARY OF THE INVENTION 
     An object of this invention is to provide a carcinostatic agent having an immunopotentiating carcinostatic effect which contains as an active ingredient Eisenin (tripeptide) of the general formula: 
     
         L-Pyroglu-L-Gln-L-Ala 
    
     wherein Pyroglu, Gln and Ala represent pyroglutamic acid, glutamine and alanine, respectively. 
     Eisenin is a tripeptide extracted from seaweed Eisenia bicyclis, and it has lately become possible to synthesize it. It is colorless long needles having a silk-thread gloss, positive in Biuret reaction, shows somewhat shrinkage at a melting point of 180° C. and decomposes on melting at 225°-226° C., and its aqueous solution is acidic. This invention has been accomplished based on a new development of Eisenin as a carcinostatic agent having an immunopotentiating carcinostatic effect. 
     In particular, the anti-tumor effect of Eisenin in accordance with this invention is not a direct effect but its effect is manifested in such way that non-specific immunological ability of the vital body is increased, and since Eisenin itself has a simple tripeptide structure, its production etc. is easy. Further, since Eisenin is crystalline, it may be formed into fine powder, and since it is water soluble, it may be processed into various pharmaceutical forms such as injections, tablets, ointments and suppositories using pharmaceutically acceptable carriers and excipients commonly employed in the pharmaceutical practice. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a graph showing the anti-tumor effect of Eisenin against the isologous allogenic tumor; 
     FIG. 2 is a graph showing the life prolonging effect of the same; 
     FIG. 3 is a graph showing the anti-tumor effect where the T-cells do not participate; 
     FIG. 4 is a graph showing the life prolonging effect of the same; and 
     FIG. 5 is a graph showing the anti-tumor effect of Eisenin against the isologous xenogenic tumor. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     As the result of the present inventor&#39;s study on Eisenin obtained by extraction from seaweed Eisenin bicyclis or synthesis, it has now been found that this is effective as a carcinostatic agent having an immunopotentiating carcinostatic effect. The results of animal experiments etc. conducted on the immunopotentiating carcinostatic effect of Eisenin are described hereinbelow. In the figures and tables, the legends Eisenin Treated and Non-treated means Eisenin administered and not administered respectively. 
     (1) Animal Experiments 
     The animals used as experimental materials were Balb/C mice and nude mice, and the tumors used were Colon (Balb/C Colon cancer) and Sarcoma 180. 
     a. 1,000,000 Cells of Colon 26 were transplanted subcutaneously to Balb/C mice. Eisenin was intraperitoneally administered to the mice at a rate of 5 mg per animal every other day three times, and the weight of the tumor and the survival rate were examined. The results thereof are set forth in FIG. 1 and FIG. 2. 
     b. 1,000,000 Cells of Colon 26 were transplanted subcutaneously to nude mice, Eisenin was intraperitoneally administered to the mice at a rate of 5 mg per animal every other day twice, and the weight of the tumor and the survival rate were examined. The results are set forth in FIG. 3 and FIG. 4. 
     c. 1,000,000 Cells of Sarcoma 180 were transplanted subcutaneously to Balb/C mice, Eisenin was intraperitoneally and orally administered to the mice at a rate of 5 mg per animal every other day three times, and the weight of the tumor was examined. The results are set forth in FIG. 5. 
     d. 10 6  Cells of sheep red blood cells (S R bc) were intravenously injected to Balb/C mice, four days later, 10 8  cells of sheep red cells and phosphate buffer solution (PBS) were subcutaneously injected to the sole of the left foot and at the same time the phosphate buffer solution (PBS) alone was subcutaneously injected to the sole of the right foot, and two days later, the thickness of both feet were measured to examine the delayed hyper-sensitivity. The results are set forth in Table 1. 
     
                                           TABLE 1__________________________________________________________________________Delayed Hyper-sensitivity__________________________________________________________________________Group 1 T (+). E (+)  Group 2 T (+). E (-) L   R   L - R         ##STR1##                  L   R   L - R                               ##STR2##__________________________________________________________________________1.59    1.57   0.02 1        1.91                     1.69                         0.22 131.56    1.54   0.02 1        1.83                     1.83                         0    01.87    1.68   0.19 11       1.91                     1.80                         0.11 61.95    1.49   0.46 31       1.86                     1.67                         0.19 111.75    1.67   0.08 5        1.78                     1.69                         0.09 51.54    1.57   0.03 0        1.96                     1.72                         0.24 141.58    1.54   0.04 3        1.71                     1.52                         0.19 131.90    1.65   0.25 15       1.82                     1.69                         0.13 8--  --  --   --       1.83                     1.87                         0.04 0--  --  --   --       1.77                     1.77                         0    0Average      8        Average      7__________________________________________________________________________Group 3 T (-). E (-)  Group 4 T (-). E (+) L   R   L - R         ##STR3##                  L   R   L - R                               ##STR4##__________________________________________________________________________1.90    1.85   0.05 3        2.09                     1.86                         0.23 121.95    1.85   0.10 5        2.18                     1.98                         0.20 102.08    1.92   0.16 8        1.84                     1.86                         0.02 01.89    1.78   0.11 6        1.93                     1.81                         0.12 71.91    1.82   0.09 5        1.87                     1.72                         0.15 92.01    1.78   0.23 13       1.93                     1.61                         0.32 201.97    1.91   0.06 3        1.87                     1.88                         0.01 01.99    1.80   0.19 11       1.93                     1.87                         0.06 31.92    1.82   0.10 5        --  --  --   --1.92    1.75   0.17 10       --  --  --   --Average      7        Average      8__________________________________________________________________________ Notes: 1.T (+) means tumor transplanted; T (-) means tumor not transplanted. 2. E (+) means Eisenin treated; E (-) means Eisenin nontreated. 3. L means the thickness of the left foot (mm); R means the thickness of the right foot (mm). 
    
     (2) In Vitro Assay 
     a. Sensitivity Test 
     Colon 26 was tissue cultured, and brought into contact with Eisenin at various concentrations for 3 days, tritium thymidine, uridine and leucine were added respectively, and the inclusion into the cells was examined to determine the inhibition index. The results of the examination on the influence of Eisenin on the syntheses of DNA, RNA and protein are set forth in Table 2. 
     
                       TABLE 2______________________________________Sensitivity TestCon-centrationof Eisenin                                 Con-(mg/ml)  1.0     0.1     0.01  0.001 0.0001                                      trol______________________________________Counts per    140430  190120  207474                          220010                                231236                                      218622Minute inthe Case of    171170  203872  229569                          213816                                219336                                      220775TritiumThymidine    163554  201151  240628                          221170                                224786                                      245962(CPM)    176167  223845  246273                          213017                                233612                                      226495Average  170297  198381  238823                          218332                                229878                                      221964I.I. (%) 23      11      8     2     0Counts per    27088   32774   37043 39415 32836 32553Minute inthe Case of    20903   25271   33211 31675 29585 31801Uridine  19954   24222   31498 28671 24135 30304(CPM)    22463   26786   34408 33161 29958 27653Average  21107   25426   33039 31169 30793 31553I.I. (%) 33      19      0     0     2Counts per    6826    13311   12285 13620 12952 14674Minute inthe Case of    6150    12566   11020 14230 11936 11382Leucine  --      8076    12097 13379 11808 12690(CPM)    6587    7076    11318 11355 10952 14350Average  6521    11318   11478 13743 12232 13905I.I. (%) 53      19      17    1     12______________________________________ Notes: (1) Inhibition Index (I.I.) is calculated from the following equation, an where I.I. exceeds 75%, it is judged effective. ##STR5##-   (2) Measurement is made on a scintillation counter. 
    
     b. Cytotoxicity Test 
     Balb/C mice were intraperitoneally administered with 15 mg of Eisenin, and the control group was administered with starch. Further, the target cell employed was BW 5174 (thymoma of AKR mouse). The spleen cells and the intraperitoneal cells were respectively divided into the adherent cells and the non-adherent cells, and cytotoxicity was examined in each case. The results are set forth in Table 3. 
     
                       TABLE 3______________________________________Cytotoxicity Test            Eisenin  Control            Administered                     Group______________________________________Spleen CellsTotal              24%        23%Non-adherent Cells 13%        15%(Natural Killer)Adherent Cells (Macrophage)              10%        10%Intraperitoneal CellsTotal              24%        -8%Non-adherent Cells 32%         2%(Natural Killer)Adherent Cells (Macrophage)              17%        -5%______________________________________ Notes: (1) Percent Cytotoxicity (%) in Table 3 is based on the following equation: ##STR6## (2) Measurement is made by .sup.51 Cr release assay.    (3) Acute Toxicit Test 
    
     The results obtained when 9 Balb/C mice (average body weight 20 g) were intraperitoneally administered with Eisenin are set forth in Table 4. 
     
                       TABLE 4______________________________________Acute Toxicity Test           No. of Death______________________________________Dosage of       6     7/9Eisenin(g/kg)          3     1/9______________________________________ 
    
     To summarize the results of the assays described above, as demonstrated in the results in Table 1 and Table 2, in the case of such isologous allogenic tumor as Colon 26 against Balb/C mice, there is observed a significant difference between the Eisenin treated group and the Eisenin non-treated group, thus an anti-tumor effect of Eisenin is recognized; further as shown in FIGS. 3 and 4, also in the case of the nude mice free from T cells, similar results have been obtained; still further as shown in FIG. 5, also in the case of such isologous xenogenic tumor as Sarcoma 180 against Balb/C mice, a similar anti-tumor effect is recognized. On the contrary, as can be seen from the results of the delayed hyper-sensitivity test on Balb/C mice shown in Table 1, there is hardly any difference between the the average values of the four groups of combinations of the presence and absence of tumor transplantation and the presence and absence of treatment with Eisenin, and therefore Eisenin has no effect to activate the immunological action in which T cells participate, and also from the sensitivity test results shown in Table 2, the inhibition index (I.I.) is low in any case, which indicates no inhibition effect by Eisenin, that is, it may be recognized that there is no direct effect of Eisenin on tumor cells. In addition, in the results of the cytotoxicity test shown in Table 3, although there is no significant difference in the case of the spleen cells, there is a significant difference in the case of the intraperitoneal cells, and as the result, it can be recognized that the immunity imparting effect by Eisenin is mainly due to the activation of the adherent cells and the non-adherent cells in the peritoneal cavity. Furthermore, according to the acute toxicity test, the LD 50  of Eisenin is in the vicinity of 5 g/kg, and the effective dosage as estimated from the above various test results is suitably about  15 mg per 20 g based on the average body weight of Balb/C mice used in the experiments taken as 20 g, that is, about 75 mg/kg, and it is believed appropriate to administer this dosage in several doses.