Abstract:
A pharmaceutical composition that inhibits the growth of tumors and cancers in mammals and can be used to treat viral infections that comprises a fungicide is disclosed. The particular fungicide used is a benzimidazole derivative.

Description:
This is a division of application Ser. No. 08/771,193, filed on Dec. 20, 1996, now U.S. Pat. No. 5,767,138. 
    
    
     TECHNICAL FIELD 
     This invention is a pharmaceutical composition that inhibits the growth of cancers and tumors in mammals, particularly in human and warm blooded animals. It is also effective against viruses and can be used to treat viral infections. The composition contains a benzimidazole derivative. 
     BACKGROUND OF THE INVENTION 
     Cancers are the leading cause of death in animals and humans. The exact cause of cancer is not known, but links between certain activities such as smoking or exposure to carcinogens and the incidence of certain types of cancers and tumors has been shown by a number of researchers. 
     Many types of chemotherapeutic agents have been shown to be effective against cancers and tumor cells, but not all types of cancers and tumors respond to these agents. Unfortunately, many of these agents also destroy normal cells. The exact mechanism for the action of these chemotherapeutic agents are not always known. 
     Despite advances in the field of cancer treatment the leading therapies to date are surgery, radiation and chemotherapy. Chemotherapeutic approaches are said to fight cancers that are metastasized or ones that are particularly aggressive. Such cytocidal or cytostatic agents work best on cancers with large growth factors, i.e., ones whose cells are rapidly dividing. To date, hormones, in particular estrogen, progesterone and testosterone, and some antibiotics produced by a variety of microbes, alkylating agents, and anti-metabolites form the bulk of therapies available to oncologists. Ideally cytotoxic agents that have specificity for cancer and tumor cells while not affecting normal cells would be extremely desirable. Unfortunately, none have been found and instead agents which target especially rapidly dividing cells (both tumor and normal) have been used. 
     Clearly, the development of materials that would target tumor cells due to some unique specificity for them would be a breakthrough. Alternatively, materials that were cytotoxic to tumor cells while exerting mild effects on normal cells would be desirable. Therefore, it is an object of this invention to provide a pharmaceutical composition that is effective in inhibiting the growth of tumors and cancers in mammals with mild or no effects on normal cells. 
     More specifically, it is an object of this invention to provide an anti-cancer composition comprising a pharmaceutical carrier and a benzimidazole derivative as defined herein along with a method for treating such cancers. 
     These compositions are also effective against viruses and can be used to treat viral infections. Therefore it is another object of this invention to provide a method of treating viral infections such as HIV, influenza and rhinoviruses. 
     These and other objects will become evident from the following detailed description of this inventions. 
     SUMMARY OF THE INVENTION 
     A pharmaceutical composition for treatment of mammals, and in particular, warm blooded animals and humans, comprising a pharmaceutical carrier and an effective amount anti-cancer compound selected from the group consisting of: ##STR1## wherein X is hydrogen, halogen, alkyl of less than 7 carbon atoms or alkoxy of less than 7 carbon atoms; n is a positive integer of less than 4; Y is hydrogen, chlorine, nitro, methyl or ethyl; and R is hydrogen, or an alkyl group of from 1 to 8 carbon atoms and R 2  is 4-thiazolyl, NHCOOR 1  wherein R 1  is aliphatic hydrocarbon of less than 7 carbon atoms, and preferably an alkyl group of less than 7 carbon atoms is claimed. Preferably the compositions are: ##STR2## wherein R is an alkyl of 1 through 8 carbon atoms and R 2  is selected from the group consisting of 4-thiazolyl, NHCOOR 1 , wherein R 1  is methyl, ethyl or isopropyl and the non-toxic, pharmaceutically acceptable acid addition salts with both organic and inorganic acids. The most preferred compounds are 2-(4-thiazolyl)benzimidazole, methyl-(butylcarbamoyl)-2-benzimidazolecarbamate and 2-methoxycarbonylamino-benzimidazole and those wherein Y is chloro. 
     These compositions can be used to inhibit the growth of cancers and other tumors in humans or animals by administration of an effective amount either orally, rectally, topically or parenterally, intravenously or by injection into the tumor. These compositions do not significantly affect healthy cells as, compared to adriamycin which has a detrimental effect on healthy cells. 
     DETAILED DESCRIPTION OF THE INVENTION 
     A. Definitions 
     As used herein, the term &#34;comprising&#34; means various components can be conjointly employed in the pharmaceutical compositon of this invention. Accordingly, the terms &#34;consisting essentially of&#34; and &#34;consisting of&#34; are embodied in the term comprising. 
     As used herein, a &#34;pharmaceutically acceptable&#34; component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. 
     As used herein, the term &#34;safe and effective amount&#34; refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. The specific &#34;safe and effective amount&#34; will, obviously, vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. 
     As used herein, a &#34;pharmaceutical addition salts&#34; is salt of the anti-cancer compound with an organic or inorganic acid. These preferred acid addition salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. 
     As used herein, a &#34;pharmaceutical carrier&#34; is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the anti-cancer agent to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. 
     As used herein, &#34;cancer&#34; refers, to all types of cancers or neoplasm or malignant tumors found in mammals. 
     As used herein, the &#34;anticancer compounds&#34; are the benzimidazoles, and their salts. The exact benzimidazoles are described in detail below. The preferred materials are the products sold under the names &#34;thiabendazole®&#34;, &#34;benomyl®&#34; and &#34;carbendazim®&#34; by BASF and Hoechst, DuPont and MSD-AgVet. 
     As used herein &#34;viruses&#34; includes viruses which infect animals or mammals, including humans. Viruses includes HIV, influenza, polio viruses, herpes, rhinoviruses, and the like. 
     B. The Anti-Cancer Compounds 
     The anti-cancer compounds are benzimidazole derivatives which are known for their antifungal activities. They are systemic fungicides used to prevent and eradicate fungi. The compounds have the following structure: ##STR3## wherein X is hydrogen, halogen, alkyl of less than 7 carbon atoms or alkoxy of less than 7 carbon atoms; n is a positive integer of less than 4; Y is hydrogen, chlorine, nitro, methyl or ethyl; and R is hydrogen or an alkyl group having from 1 to 8 carbons, and R 2  is 4-thiazolyl, NHCOOR 1  wherein R 1  is aliphatic hydrocarbon of less than 7 carbon atoms, and preferably and alkyl group of less than 7 carbon atoms. Preferably the compositions are: ##STR4## wherein R is an alkyl of 1 through 8 carbon atoms and R 2  is selected from the group consisting of 4-thiazolyl, NHCOOR 1 , wherein R 1  is methyl, ethyl or isopropyl and the non-toxic, pharmaceutically acceptable acid addition salts with both organic and inorganic acids. 
     The most preferred compounds are 2-(4-thiazolyl)benzimidazole, methyl -(butylcarbamoyl)-2-benzimidazolecarbamate and 2-methoxycarbonylamino-benzimidazole and the compounds wherein Y is chloro and X is hydrogen. 
     These compounds are prepared according to the method described in U.S. Pat. No. 3,738,995 issued to Adams et al, Jun. 12, 1973. The thiazolyl derivatives are prepared according to the method described in Brown et al., J. Am. Chem. Soc., 83 1764 (1961) and Grenda et al., J. Org. Chem., 30, 259 (1965). 
     It is believed that fungicides, particularly systemic fungicides, have the capability of reducing tumors or decreasing their growth significantly. Systemic fungicides have the ability to migrate through the plant or animal body. While this is a positive attribute, it is not an essential requirement of the effective compounds for treating viral infections, cancers or tumors. 
     C. Dosage 
     Any suitable dosage may be given in the method of the invention. The type of compound and-the carrier and the amount will vary widely depending on the species of the warm blooded animal or human, body weight, and tumor being treated. Generally a dosage of between about 2 milligrams (mg) per kilogram (kg) of body weight and about 400 mg per kg of body weight is suitable. Preferably from 15 mg to about 150 mg/kg of body weight is used. Generally, the dosage in man is lower than for small warm blooded mammals such as mice. A dosage unit may comprise a single compound or mixtures thereof with other compounds or other cancer inhibiting compounds. The dosage unit can also comprise diluents, extenders, carriers and the like. The unit may be in solid or gel form such as pills, tablets, capsules and the like or in liquid form suitable for oral, rectal, topical, intravenous injection or parenteral administration or injection into or around the tumor. 
     D. Dosage Delivery Forms 
     The anti-cancer compounds are typically mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used. The active agent can be coadministered in the form of a tablet or capsule, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms would also include minerals and other materials to make them compatible with the type of injection or delivery system chosen. 
    
    
     Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker &amp; Rhodes, Editors, 1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976). 
     E. Method of Treatment 
     The method of treatment can be any suitable method which is effective in the treatment of the particular virus or tumor type that is being treated. Treatment may be oral, rectal, topical, parenteral or intravenous administration or by injection into the tumor and the like. The method of applying an effective amount also varies depending on the tumor being treated. It is believed that parenteral treatment by intravenous, subcutaneous, or intramuscular application of the benzimodale compounds, formulated with an appropriate carrier, additional cancer inhibiting compound or compounds or diluent to facilitate application will be the preferred method of administering the compounds to warm blooded animals. 
     The method of treating viral infections may also be by oral, rectal, topical, parenteral or intravenous administration. The actual time and dosage will depend on the virus being treated and the desired blood levels. 
     The following examples are illustrative and are not meant to be limiting to the invention. 
     Colon, Breast and Lung Tumor Cells Test 
     The following cell culture tests were performed to test the toxicity of the benzimidazole compounds on colon, breast and lung human tumor cells. The viability of the cells were tested by looking at MTT (3- 4,5-dimethylthiazol-2-yl!-2,5-diphenyltetrazolium bromide) reduction. MTT assay is a well known measure of cell viability. 
     The colon tumor cells (HT29 from American Type Culture Collection (ATCC) ) and the breast cells (MX1 from cell lines from ATCC) were cultured in Eagle&#39;s Miminal Essential Medium with 10% fetal bovine serum. The lung tumor cells (A549 from ATCC cell lines) were cultured in Ham&#39;s F12 medium with 10% fetal bovine serum. 
     The tumor cells were passaged and seeded into culture flasks at the desired cell densities. The culture medium was decanted and the cell sheets were washed twice with phosphate buffered saline (PBS). The cells were trypsinized and triturated prior to seeding the flasks. Unless otherwise indicated the cultures were incubated at 37°±1° C. in a humidified atmosphere of 5±1% carbon dioxide in air. The cultures were incubated until they were 50-80% confluent. 
     The cells were subcultured when the flasks were subconfluent. The medium was aspirated from the flasks and the cell sheets rinsed twice with PBS. Next, the Trypsin Solution was added to each flask to cover the cell sheet. The Trypsin Solution was removed after 30-60 seconds and the flasks were incubated at room temperature for two to six minutes. When 90% of the cells became dislodged, growth medium was added. The cells were removed by trituration and transferred to a sterile centrifuge tube. The concentration of cells in the suspension was determined, and an appropriate dilution was made to obtain a density of 5000 cells/ml. The cells were subcultured into the designated wells of the 96-well bioassay plates (200 microliter cell suspension per well). PBS was added to all the remaining wells to maintain humidity. The plates were then incubated overnight before test article treatment. 
     Each dose of test article was tested by treating quadruplicate wells of cultures with 100 microliter of each dilution. Those wells designated as solvent controls received an additional 100 microliter of methanol control; negative controls wells received an additional 100 microliters of treatment medium. PBS was added to the remaining wells not treated with test article or medium. The plates were then incubated for approximately 5 days. 
     At the end of the 5 day incubation, each dose group was examined microscopically to assess toxicity. A 0.5 mg/ml dilution of MTT was made in treatment medium, and the dilution was filtered through a 0, 45 micrometer filter to remove undissolved crystals. The medium was decanted from the wells of the bioassy plates. Immediately thereafter, 2000 microliter of the filtered MTT solution was added to all test wells except for the two untreated blank test wells. The two blank wells received 200 microliters of treatment medium. The plates were returned to the incubator for about 3 hours. After incubation, the MTT containing medium was decanted.. Excess medium-was added to each well and the plates were shaken at room temperature for about 2 hours. 
     The absorbance at 550 nm (OD 550 ) of each well was measured with a Molecular Devices (Menlo Park, Calif.) VMax plate reader. 
     The mean OD 550  of the solvent control wells and that of each test article dilution, and that of each of the blank wells and the positive control were calculated. The mean OD 550  of the blank wells was subtracted from the mean of the solvent control wells, and test article wells, respectively to give the corresponding mean OD 550 . ##EQU1## 
     Dose response curves were prepared as semi-log plots with % of control on the ordinate (linear) and the test article concentration on the abscissa (logarithmic). The EC 50  was interpolated from the plots for each test article. 
     For the test articles administered in methanol, separate responses were prepared to correct for the methanol data. 
     Adriamycin was used as a positive control. In all cases, it was more toxic than any of the test materials by one or two logs. Adriamycin is one of the more potent agents in current use and one with significant side effects. The peak plasma concentration of other, quite effective chemotherapeutic agents may be 10 to 50 times higher than that of Adriamycin. 
     The EC 50  is the concentration at which one half of the cells are killed. 
     
                       TABLE 1______________________________________  EC-50 Result (ppm)Test Material    HT29   HT29     MX1  MX1    A549 A549______________________________________Adriamycin    0.03   0.006    0.02 0.001  0.03 0.009benomyl  0.742  0.747    1.42 2.42   0.980                                     1.02carbendazim    0.621  0.662    0.829                         0.856  0.856                                     0.836______________________________________ 
    
     In normal healthy cells, the following results were obtained. As is evident, the benomyl and carbendazim were much less toxic to normal healthy cells than adriamycin. 
     
                       TABLE 2______________________________________EC-50   Broncheal   KerotinoyleTest Material   Cells       Cells        Fibroblasts______________________________________Benomyl 0.728   0.682   3.26   2.4   3.24   2.81Carbendazin   0.320   0.506   0.752  0.822 1.52   1.42Adriamycin   0.015    0.0020  0.0035                           0.0093                                 0.065 0.10______________________________________ 
    
     In a related study using lung tumor cells (A-549) breast tumor cells (MCF-7) and colon tumor cells (HT-29), thiabendazol, a systemic fungicide, effectively killed these cells. Table 3 summarizes the results 
     
                       TABLE 3______________________________________      Optical DensityConcentration (ppm)        A-549       MCF-7   HT-29______________________________________0-Control    0.600       0.245   0.398173          0.007       0.007   0.00535           0.411       0.025   0.01117.3         0.851       0.258   0.2043.46         1.12        0.466   0.7130.87         1.32        0.507   0.852______________________________________ 
    
     These experiments show that these compositions are effective in killing tumor cells. 
     These same systemic fungicides are effective against viruses including HIV, influenza, rhionoviruses and herpes viruses. The fungicides can be used alone or in combination with other fungicides.