Abstract:
This invention is concerned with clinical investigation of therapeutic effects of garlic extracts containing allicin, diallyl disulfide, and diallyl trisulfide. Because the intake of garlic or therapeutically active substance such as allicin from garlic, induces immunostimulation and possess strong antitumor effects concomitant with apoptosis of tumors that can in turn induces a programmed cell death of human prostatic and bladder tumor cells. This invention claims that garlic and/or garlic extracts possess very effective therapeutic substances not only to treat human prostate and bladder cancers to extend life span of patients, but also to suppress or prevent bladder and prostate gland cancers.

Description:
BACKGROUND OF THE NVENTION  
         [0001]    1. Field of the Invention  
           [0002]    This invention is related to new therapeutic application of garlic extracts containing allicin, diallyl disulfide, trisulfide, etc. more specifically against human prostate gland and bladder tumors. This therapeutic application relates to strong immunostimulation mediated anticancer effects elicited by allicin, diallyl disulfide, and diallyl trisulfide in garlic extracts, and subsequently induces programmed prostate and bladder cancer cell death.  
           [0003]    2. Description of the Prior Art  
           [0004]    Prostate cancer death in the U.S and Europe is the most prevalent and the second highest cancer death among male population. As of aged-male populations increases and importation of advanced diagnostic techinique for prostate cancer in Korea, together with increased consumption of dairy products, and a dramatic changes in dietary habits are associated closely with increase in male prostate cancer patients and increase in male mortality due to prostate cancers. Thus, prostate cancers in Korea is expected to be one of the major socially important disease.  
           [0005]    Unfortunately, when many of the patients are diagnosed as a prostate cancers, prostate tumors are already metastasized to bone marrow, and lymph nodes and such cases, only treatment mode left is hormonal therapy (either radical orchiectomy of both testes and/or continued injection of LHRH analogues) to which, most of the patients respond well to primary treatment, however, more than half of the patients develop resistance to hormonal treatment and unfortunately, these patients die within one year to a year and a half. Consequently, it was very much desired to find a new therapeutic regimens or increase quality of life for patients, who are resistant to hormonal therapy, such effective new therapeutic regimens have remained to be discovered. Furthermore, if it is feasible to find a preventive therapeutic agents for prostate cancers without a serious side effects, the incidence of prostatic cancers will be signjficantly reduced and discovery of such therapeutic agents will be highly desired.  
           [0006]    The etiology of malignant bladder cancers are attributed to a long term exposure to cigarette smoke and/or environmental carcinogens. In Korea, bladder cancers are the highest among urinary, and reproductive tract cancers and bladder cancers in male ranks the top 5th of all male cancer categories. Male bladder cancer incidence is expected to increase and likewise, mortality rate is expected to increase. Bladder cancers are classified into 3 categories based on progression of bladder cancers to superficial, invasive, and metastatic cancers. Incidence of superficial bladder cancer is the highest and even if this class of bladder cancer is found at an early stage, resection of urethra and bladder are required and even if all visible tumors are removed by resection, the recurrence rate is still as high as 50˜70%. The repeated resection lead to a higher incidence of complication such as urethra stricture, bladder perforation, and a severe inflammation. In recent times, in attempt to reduce recurrence of bladder tumors, very often institute chemotherapeutic agents or immunomodulators following the initial tumor resection. Nonetheless, extreme bloody urine, and inflammation of urethra ensu further complicates and cannot stop the continued progression of bladder cancers. Furthermore, in case of invasive bladder cancers, a radical resection of bladder is required and the use of an artificial bladder is required and cause not only severe inconvenience of daily livelihood of patients, but also often leads to postsurgical complications.  
           [0007]    When patients are diagnosed as metastasized bladder cancers, only treatment course left is to treat the patients with aggressive chemotherapy, but leads to severe complications. Despite the aggressive chemotherapy, the prognosis of patients are not so good. For this reason, it is a vital importance that more effective therapeutic agents or highly effective preventive agents against occurrence of bladder cancers need to be developed and to be used effectively to deal with human bladder cancers. Especially, it is highly desirable to develop such effective chemopreventive agents without severe side effects or unreasonable cost.  
         SUMMARY OF THE INVENTION  
         [0008]    These inventors sought to find a new alternative therapeutic or preventive agents from either therapeutic chemicals or the extracts of natural plants against both prostatic and bladder cancers by selectively eradicating both prostatic and bladder cancer cells. We were able to discover and complete our invention as a result of this experimental research that garlic extracts clearly demonstrated that not only the it possesses chemopreventive effects, but also ti induced programmed cancer cell death of both prostate and bladder cancer cells.  
           [0009]    Furthermore, the objective of the invention is to propose that garlic extracts containing allicin is highly effective preventive and/or chemotherapeutic agent(s) against prostatic and bladder cancers. The other objective of this invention is that a composition of garlic extracts containing allicin is very effective chemotherapeutic and chemotherapeutic agents against prostatic and bladder cancers.  
           [0010]    The objective of the invention described in the preceding is accomplished by experimental confirmation that garlic extracts containing allicin, diallyl disulfide, diallyl trisulfide, and inclusive of other effective compounds were proven to be highly effective chemopreventive and/or chemotheraputic agents against experimental mice bearing human prostatic and bladder tumor.  
           [0011]    A chemopreventive and chemotherapeutic composition against human prostate and human bladder cancers comprises allicin-containing garlic extracts isolated and purified from garlic, as an effective ingredient. The organization of this invention is described in detail below. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0012]    The above and other objects, features and other advantageous of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:  
         [0013]    In the FIG. 1, this invention clearly demonstrates a detailed photomicrography of programmed human prostate tumor cell death after H&amp;E staining of PC-3 prostate tumor tissues following immunologically targeting human PC-3 prostate tumor bearing experimental mice models with garlic extracts containing allicin.  
         [0014]    In the FIG. 2, this invention clearly shows a detailed photomicrography human PC-3 prostate tumor cell death after processing both immunologic and tunnel assays with Apotaq Kits following immunologically targeting human tumor bearing experimental mice with garlic extracts containing allicin.  
         [0015]    In the FIG. 3, this invention clearly shows a detailed photomicrography of programmed cell death of most of the bladder tumor cells with tunnel assays with Apotaq kits 2-weeks following immunologically targeted treatment of MB-2 bladder tumor bearing mice with garlic extracts containing allicin.  
         [0016]    In the FIG. 4, this invention clearly demonstrates the evidence of therapeutic effects against bladder tumor cells by observing a programmed bladder tumor cells death evidenced by immunohistochemical and Tunnel assay with Apotaq kits after 2-weeks of continues immunologically targeting of bladder tumor bearing mice with garlic extracts with allicin. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0017]    This invention is organized in two steps: 1) describing chemotheraputic effects against prostatic tumor with garlic extracts containing allicin; 2) describing chemotherapeutic effects against bladder tumors with garlic extracts containing allicin.  
         [0018]    These inventors grounded 500kg of fresh garlic and mixed with equal amount vegetable oil and left for 4 to 6 days, then separated and purified by a column chromatography or a preparative column chromatography after removing water moisture by adding anhydrated sodium sulfate ({fraction (1/10)} of the original starting material). Finally purified garlic extracts containing allicin had a clear yellow liquid with a strong garlic odor and its specific gravity was 0.906˜0.913 and acidity was 1.0. The saponification number was 163˜180, the iodine number ranged form 94˜106, and allicin content was greater than 0.1%. The garlic extract used for the present experimental mice were tested for microbial contamination such as  E. coli , Staphylococcus, Streptococcus, and Salmonella repeatedly prior to animal treatment. In addition, garlic extracts were tested for heavy metals as well as pesticide contaminants such as BHC, DDT, Aldrin, Dieldrin, and Dieldrin. The garlic extracts with contaminant heavy metals less than 30 ppm, and pesticides residue contaminant levels less than 0.01 ppm were only used.  
         [0019]    Concentrations of allicin in garlic extracts can be adjusted by adding the appropriate amount of pharmaceutical formulation used for antibacterial use, if necessary, to optimize the formulation, frequency of dosing, and interval of dosing for the treatment of prostatic or bladder cancer patients and the extent of adjustment were based on patients age, body weight, the clinical status of cancers, and general health.  
         [0020]    The formulation of garlic extract containing allicin as described technically in the preceding statements is directly administered using syringes into prostatic and bladder tumors as guided by CT scan, MRI or biosonargram imaging. Direct administration of allicin into tumor masses shows not only induce programmed tumor cell death, and inhibit tumor cell growth, but also tumor regression by activation of immune functions. Furthermore, this kind of therapeutic actions are attributed to interactions between cancer-cell antigens and allicin as a biological response modfier to interact by either direct or indirect action on immune systems. Furthermore, either prostatic and bladder tumor growth rates are closely dependent on angiogenesis, and if angiogenesis is inhibited effectively, prostatic and bladder tumor growth will be effectively inhibited. Even among the normal healthy subjects, who have higher risk of prostate cancers due to aging and race (black), family-linked high prostatic tumor risk may be subjected to prophylactic administration of garlic extracts containing allicin directly adjacent to prostate glands and thereby potentially preventing protstatic tumors. This injection procedure of garlic extracts containing allicin to adjacent areas of prostate gland can be easily performed in the ambulatory care unit without any side effects such as bleeding or pain. Administration of garlic extract containing allicin will interact with cells adjacent to prostate glands and also modulate immune functions to prevent prostatic tumor formation by blocking transformation process of normal prostatic cells to prostatic tumor cells.  
         [0021]    Likewise, a direct administration of garlic extracts containing allicin as a cancer preventive agent can effectively inhibit bladder cancer development among the aged high risk adult males and females with a long term smoking habits or among the high risk occupational group working in the leather industry without a sginificant side effects such as hemorrhage or pain.  
         [0022]    A possible mechanism(s) of cancer preventive action is as already described earlier.  
         [0023]    A direct administration to target tumors with garlic extracts containing allicin as a new chemotherapeutic agents to treat prostatic and bladder cancers may be highly effective a partly due to anti-angiogenesis, inhibiting the growth of microvessels that supplies the essential nutritions to growing tumor tissues. A direct aministration of garlic extracts containing alicin into vessels of tumors can possibly inhibit the growth of microvessls to tumor tissue masses. Jugular vein angiography can easily monitor vessel size, location, and vessel numbers in tumors using a microvessel angiography during the direct injection of garlic extracts containing allicin into prostatic or bladder cancer patients to possibly attain highly effective therapeutic response without any serious side effects of normal tissue adjacent to that of tumor tissues. Furthermore, direct administration of garlic extracts containing allicin directly into tumor vessels can directly regulate a local cellular immunologic response along with activation of humoral immune response, which in turn immediately leads to programmed endothelial cell deaths in both prostatic and bladder tumor masses and thereby, block tumor growth.  
         [0024]    Garlic is a widely used food in general and not only price is cheap but unlike most of drugs, garlic extracts containing allicin does not cause any side effects. Thus, it is an ideal for a continuous theraphy by oral route and expected of continued maintenance of stimulated immune response and with consequent prevention of prostatic and bladder tumor formations. For oral route formulation of garlic extracts containing allicin is similar to that of other oral drug formulations. It is also possible to formulate to preserve the stability of allicin since continuous oral administration is very effective mode of preventing prostatic and bladder tumor formations. This is one of the key content of invention.  
         [0025]    For this invention related investigation of garlic extracts containing allicin against prostatic tumor animal models, the test garlic extracts used were without heavy metals, pesticide, BHC, DDT, Aldrin, Dieldrin and Endrin or microbial contaminations, general bacteria,  E. coli , Stretococci, Staphyosocci, and Salmonella, etc.  
         [0026]    This invention relates to garlic extracts can be mixed with binding inert materials to manufacture the capsules, beads, and tablets.  
         [0027]    This invention deals with a detaild description of organization and the functional effects of an example will be presented in the below to describe its purpose, and not to limit the rights of the invention within the experimental results.  
         [0028]    Example 1: Investigation of anticancer effects against human prostate tumors.  
         [0029]    To establish human prostatic tumor animal models, 1×106 hormone-independent PC-3 human prostatic tumor cells grown in T-medium were inoculated on both side of abdoman of athymic nude mice (nu/nu). Experimental animal groups consisted of the treatment and control group(s). The treatment group (I) was given direct injection of garlic extracts containing allicin into the tumor transplated site(s). The treatment group consisted of 15 mice with total of 30 prostatic tumor sites and the day after the tumor transplantation, once weekly for total of 5 week duration with each treatment contained 5 mg garlic extract containing 0.03 mg allicin (the total dose received was per mouse 25 mg garlic extracts containing 0.15 allicin). In contrast, the control group (II) consisted of 8 athymic nude mice and directly injected into tumor sites with saline. Tumor sizes were measured weekly interval using a tumor caliper. In the treatment group, allicin modulated immunotherapeutic effects were evaluated with respect to tumor incidence, tumor growth rates, histopathologic evaluation and the duration of animal survival time and if wanted statistically analyzed to determine any significant differences between the control and the treated.  
         [0030]    As shown in Table 1, the day after xenografting of human prostatic tumor cell lines, weekly immunotherapy with garlic extracts with allicin for 5 weeks in the experimental group I, showed only 13.3% incidence of prostatic tumors. Thus, 5 week immunotheray with garlic extracts were statistically(fishers exact test) significant (p&lt;0.01) in the test group as compared to that of controls. Furthermore, the tumor sizes and the volumes in the treatment group I are significantly reduced as compared to that of the controls group II by Mann-Whitney U nonparametric test (P&lt;0.01). The results clearly demostrated that immunotherapy with garlic extracts containing allicin using the athymic nude mice bearing human prostatic tumor cells effectively prevented tumor appearance, tumor growth and progression.  
                             TABLE 1                           The result of immunotherapeutic effects of garlic extracts containing       allicin in athymic nude mice xenograted with hormone-independent       human prostatic tumor cell lines (PC-3)                Prostatic tumor incidence   Prostatic tumor sizes       Experimental groups   (day 21)   (day 35, mm ± SD)               Group I   4/30 (13.3%)    190.25 ± 68.89       (Treatment group)       Group II   8/8 (100.0%)   1209.75 ± 217.1       (Saline control group)                  
 
         [0031]    As shown in Tables 1 &amp; 2, Gross observation of tumor incidence, tumor sizes, and growth rates was also confirmed by microscopic histopathology as well as immunohistochemistry. These results show that immunotherapeutic targetting with garlic extracts containing allicin to prostatic cancers are proven to be very effective ways to induce programmed prostatic tumor cell deaths.  
         [0032]    Example 2: Investigation of anticancer effects against human bladder cancers.  
         [0033]    Experimental example 1  
         [0034]    To evaluate anticancer effects against human bladder tumors, 1×105 mouse transitional epithelial bladder tumor cell lines, MBT-2 cells were transplanted on the abdominal region of 24 female C3H/He mouse strain. The experimental mice were split into two groups, each consisted of 12 mice. A day after tumor cell inoculation, the experimental group  1  were treated directly into tumor transplafation site(s) with 5 mg garlic extracts containing 0.03 mg allicin (total dose received: 25 mg garlic extracts cotaining 0.15 mg allicin) per weekly for the total duartion of 5 weeks. The mice in the experimental group  2  was given weekly of saline and served as a control experimental group. Tumor growth and sizes were determined weekly using a tumor caliper. Immunotherapeutic effects of allicin was evaluated by tumor incidence, tumor growth rates, histopathology of tumors, and the survival rates of tumor bearing mice and the final results were statiscally analyzed.  
         [0035]    As shown in the Table 2, the results demonstrate that after 5 weeks of treatment with garlic extracts containing allicin in the experiment group  1  that only  2  out 12 mice showed bladder tumors (16.7%), while all control mice in the experimental group  2  showed the tumor growth (100%). Thus, the results clearly demonstrates the chemopreventive effects of garlic extracts containing allicin significantly reduced the incidence of bladder tumors and the Fishers exact test showed a significant difference between the control and the test groups (p&lt;0.05). Futhermore, the mice in the experimental group  1  treated with garlic extracts containing allicin for the 5-week duration did not result in any animal death due to treatment related toxicity.  
                             TABLE 2                           The result of prevention of bladder tumor formation by imunotherapeutic       effects of garlic extracts cotaining allicin in MBT-2 mouse bladder       tumor bearing mice                    Survival of mice           Bladder tumor incidence   (After treatment of       Experimental groups   (Day 28)   garlic extracts)               Group I    2/12 (16.7%)   0/12 (0.0%)       Z(Treatment group)       Group II   12/12 (100%)   0/12 (0.0%)       (Saline control group)                  
 
         [0036]    Experimental Example 2  
         [0037]    Another experiment that consisted of 24 female C3H/He mouse strain were transplanted with 1×105 cultured transitional bladder epithelial cell tumors, MBT-2 cell lines. Seven days after tumor transplants, when bladder tumor sizes ranged 55-65 mm3 in all 24 mice, the mice were divided into two experimental groups, each consisted of 12 mice each. The mice in experimental group  1  was treated weekly for the 5-weeks duration with 5 mg of garlic extracts containing 0.03 mg allicin by directly injeeting into tumors sites (Total dose: 25 mg garlic extracts containing 0.15 mg allicin), while the mice in the experimental 2 was treated same way wtih saline. Subsequently, the tumor sizes were mesured weekly with a tumor caliper to assess immunotherapeutic effects of allicin with respect to tumor incidence, growth rates, hisopathological examination, and the animal survival rates. The results of the experiment in the table 3 shows that 35 days after tumor cell transplants, the average tumor volumes in the allicin treated group was 1534.8+562 mm3, however, that of the control group was 5120.6 +812.5 mm3. Therefore, it is clearly demonstrated that the average bladder tumor sizes in the allicin treatment group were significantly reduced by immunotherapeutic effects. The statistical dfferences between the treated versus that of the control was highly significant (Mann-Whitney nonprametric test, p&lt;0.05). It is clearly evident from this experiment that growth of bladder tumors these in experimental mice was significantly inhibited by garlic extracts containing allicin. Furthermore, the survival of these mice in the experimental group  1  treated with immuotherapeutic effects of garlic extracts containing allicin was significantly greater than that of control group.  
                             TABLE 3                           The result of immunotherapeutic effects of garlic extracts containing       allicin against MBT-2 bladder tumor bearing mice                Bladder tumor size(mm3)   Survival of mice       Experimental groups   (Day 35, Mean ± SD)   (Day 42)               Group I   1534.8 ± 552.4   3/12 (25%)       (Treatment group)       Group II   5120.6 ± 812.5   1/12 (8.3%)       (Saline control group)                  
 
         [0038]    After sacrificing mice with transplanted bladder tumors, the immunotherapteutic effects of garlic extracts containing allicin was evaluated by immunohistochemistry and microscopic histopathology. As hown in the FIGS. 3 and 4, it clearly demonstrated programmed cell death accompanied by tumor regression.  
         [0039]    As explained of experimental results, this invention describes and demonstrates that garlic extracts containing allicin, diallyl disulfide, diallyl trisulfide, and others, etc. possess both preventive and chemotherapeutic effects against human prostate and bladder tumors and thus, this invention is a very useful invention for biopharmaceutical industries.  
         [0040]    Although a preferred embodiment of the present invention has been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.