Method of Determining the Probability of a Therapeutic Response in Cancer Chemotherapy with Cardiac Glycoside

Abstract

A prognostic assay and kit and method of use thereof are provided. The kit and assay are used to determine the likelihood of a diseased cell or tissue having a therapeutic response to treatment with a cardiac glycoside in a disease having an etiology associated with excessive cell proliferation. The kit and assay are used to determine the ratio of isoforms of the α subunit of Na, K-ATPase obtained from the diseased cell or tissue. The kit can be used to predict the therapeutic responsiveness of cancer or tumor in a subject to treatment with a cardiac glycoside. The kit and assay can be incorporated in a method of treating a disease or disorder having an etiology associated with excessive cell proliferation with a composition comprising a cardiac glycoside.

1 ) (canceled) 
     
     
         2 ) The kit of  claim 47 , wherein the information comprises predicting that the cellular tissue will be therapeutically responsive to treatment with a cardiac glycoside if the ratio is greater than or equal to at least 1, will be at least partially therapeutically responsive to treatment with a cardiac glycoside if the ratio is within the range of 0.5 to 1.0, and/or will be substantially therapeutically non-responsive to treatment with a cardiac glycoside if the ratio is less than 0.3. 
     
     
         3 ) (canceled) 
     
     
         4 ) (canceled) 
     
     
         5 ) The of  claim 47 , wherein the information comprises predicting that diseased tissues having an α-subunit isoform ratio within the range of 1 to 100 will be more therapeutically responsive than those having an α-subunit isoform ratio less than 1 and/or predicting that those tissues with only detectable α3 isoform and no detectable α1 isoform will be the most therapeutically responsive to cardiac glycosides. 
     
     
         6 ) (canceled) 
     
     
         7 ) The kit of  claim 47 , wherein the information comprises predicting that the cellular tissue will be at least partially therapeutically responsive to treatment with a cardiac glycoside if the ratio is ≧2, will be at least partially therapeutically responsive to treatment with a cardiac glycoside if the ratio is ≧10, will be at least partially therapeutically responsive to treatment with a cardiac glycoside if the ratio is ≧25 or will be at least partially therapeutically responsive to treatment with a cardiac glycoside if the ratio is ≧75. 
     
     
         8 ) (canceled) 
     
     
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         19 ) (canceled) 
     
     
         20 ) (canceled) 
     
     
         21 ) (canceled) 
     
     
         22 ) (canceled) 
     
     
         23 ) The kit of  claim 47  comprising instruction to conduct a radiometric or densitometric analysis of a gel or Western blot assay or immunohistochemical staining assay in order to detect the presence of and quantify the content of α3 subunit isoform of Na, K-ATPase and of α1 subunit isoform of Na, K-ATPase in the sample. 
     
     
         24 ) The kit of  claim 47  comprising instruction to compare the content of α3 subunit isoform of Na, K-ATPase and of α1 subunit isoform of Na, K-ATPase in the sample relative to the content of α3 subunit isoform of Na, K-ATPase and/or of α1 subunit isoform of Na, K-ATPase in a positive control sample and/or a negative control sample. 
     
     
         25 ) The kit of  claim 47  comprising instruction to compare the content of α3 subunit isoform of Na, K-ATPase and of α1 subunit isoform of Na, K-ATPase in a tissue sample where expression of only one of α3 and α1 subunit is known to occur as a control. 
     
     
         26 ) The kit of  claim 47 , wherein the diseased cellular tissue is obtained from a subject such as a mammal. 
     
     
         27 ) The kit of  claim 26 , wherein the diseased cellular tissue is obtained from a human, cow, dog, cat, horse, pig or other domesticated animals whether of commercial value or not. 
     
     
         28 ) The kit of  claim 47 , wherein the disease or disorder having an etiology associated with excessive cell proliferation is cancer or tumor or other proliferative diseases that impacts adversely on human or animal quality of life. 
     
     
         29 ) The kit of  claim 28 , wherein the cancer or tumor is selected from the group consisting of colorectal cancer, head and neck cancer, adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, bone metastasis, sarcomas of bone, brain cancer, breast cancer, cervical cancer, non-Hodgkin's lymphoma, rectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, non small cell, small cell carcinomas, lung carcinoid tumors, malignant mesothelioma, metastatic cancer, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal cancer, nasopharyngeal cancer, neruoblastoma, neoplasms of the central nervous system, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, salivary gland cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, cancer of the ureter; uterine sarcoma, vaginal cancer, vulva cancer or Wilm's tumor. 
     
     
         30 ) The kit of  claim 28 , wherein cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, canine osteogenic sarcoma, inference human osteogenic sarcoma, human brain cancer, glioblastoma multiform and human colon cancer. 
     
     
         31 ) (canceled) 
     
     
         32 ) (canceled) 
     
     
         33 ) (canceled) 
     
     
         34 ) (canceled) 
     
     
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         36 ) The kit of  claim 47 , wherein the cardiac glycoside is present in pure form whether derived through extraction of a plant or animal source, synthesized or manufactured through chemical modification of an available cardiac glycoside. 
     
     
         37 ) The kit of  claim 47 , wherein the cardiac glycoside is present in an extract. 
     
     
         38 ) The kit of  claim 37 , wherein the cardiac glycoside extract was prepared by supercritical fluid extraction optionally in the presence of a modifier. 
     
     
         39 ) The kit of  claim 38 , wherein the SCF extract further comprises at least one other pharmacologically active agent aside from the cardiac glycoside. 
     
     
         40 ) The kit of  claim 39 , wherein the other active agent may contribute to the therapeutic efficacy of the cardiac glycoside when the extract is administered to a subject. 
     
     
         41 ) The kit of  claim 40 , wherein the other active agent functions additively or synergistically to contribute to the therapeutic efficacy of the cardiac glycoside. 
     
     
         42 ) (canceled) 
     
     
         43 ) (canceled) 
     
     
         44 ) The kit of  claim 47 , wherein the cardiac glycoside has been obtained from an oleander plant mass. 
     
     
         45 ) The kit of  claim 44 , wherein the oleander plant mass comprises  Nerium  species or of  Thevetia  species. 
     
     
         46 ) (canceled) 
     
     
         47 ) A kit suitable for use in conducting a prognostic assay, the kit comprising:
 a) a first primary antibody having a binding affinity for the α3 subunit isoform of Na, K-ATPase;   b) a second primary antibody having a binding affinity for the α1 subunit isoform of Na, K-ATPase;   c) instructions for use of kit and performance of the prognostic assay for predicting the in vivo therapeutic responsiveness of a disease or disorder, having an etiology associated with excessive cell proliferation, to treatment with a cardiac glycoside or composition comprising a cardiac glycoside; and   d) information detailing how to interpret prognostic data obtained from performance of said prognostic assay.   
     
     
         48 ) The kit of  claim 47 , wherein the kit is adapted for use in conducting a Western blot gel electrophoretic assay or the kit is adapted for conducting an immunohistochemical staining assay. 
     
     
         49 ) The kit of  claim 47  further comprising: a) a lysis composition; b) a positive control sample comprising α3 subunit isoform of Na, K-ATPase; c) a positive control sample comprising α3 subunit isoform of Na, K-ATPase and α1 subunit isoform of Na, K-ATPase; d) a negative control sample comprising α1 subunit isoform of Na, K-ATPase and excluding α3 subunit isoform of Na, K-ATPase; e) a secondary antibody, a goat anti-mouse IgG-HRP; f) gel-forming material suitable for gel electrophoretic analysis; g) radiolabeled marker; h) densitometer or radiometer; i) aqueous liquid medium; j) gel/membrane preparation kit; k) blocking solution; l) wash buffer; m) materials comprising a Western blot analysis kit; or n) a combination thereof. 
     
     
         50 ) The kit of  claim 47 , wherein the first primary antibody has specific binding affinity for the α3 subunit isoform of Na, K-ATPase. 
     
     
         51 ) The kit of  claim 47 , wherein the second primary antibody has specific binding affinity for the α1 subunit isoform of Na, K-ATPase. 
     
     
         52 ) The kit of  claim 49 , wherein the secondary antibody is Goat cc mouse IgG horse radish peroxidase or comprises other secondary antibodies from species other than mouse raised against mouse IgG with an appropriate marker attached such as horse radish peroxidase. 
     
     
         53 ) The kit of  claim 47 , wherein the primary antibodies are monoclonal antibodies. 
     
     
         54 ) The kit of  claim 49 , wherein the positive control sample is selected from the group consisting of tissue, cellular mass, cellular lysate, and membrane preparations prepared from these which can be obtained through biopsy or other means of surgical excision. 
     
     
         55 ) The kit of  claim 49 , wherein the negative control sample is selected from the group consisting of tissue, cellular mass, or cellular lysate and membrane preparations prepared from these that do not contain α3 isoform of the α-subunit of Na, K-ATPase. 
     
     
         56 ) The kit of  claim 55 , wherein the negative control comprises a cellular mass of rodent tumor tissue or mouse or rat cells grown in vitro. 
     
     
         57 ) The kit of  claim 47  comprising alternative means of determining relative Na, K-ATPase α-subunit composition and isoform ratios, wherein the alternative means comprises: use of antibodies in an enzyme linked immunoabsorbant assay or protein tissue or cell lysate array; use of Northern blot analyses and related techniques for measurement of mRNA to different Na, K-ATPase subunit isoforms; or use of immunohistochemical staining assay. 
     
     
         58 ) A method of treating, in a subject, a disease or disorder having an etiology associated with excessive cell proliferation with a composition comprising cardiac glycoside, the method comprising:
 a) using the kit of  claim 47  to determine the ratio of α3 isoform to α1 isoform of the α subunits of Na, K-ATPase in a sample obtained directly or indirectly from diseased in vivo cellular tissue of the subject with a disease or disorder having an etiology associated with excessive cell proliferation, the sample comprising one or more isoforms of the cc subunit of Na, K-ATPase; and   b) if the ratio is ≧0.3, indicating administration to the subject a composition comprising cardiac glycoside.   
     
     
         59 ) A method of treating, in a subject, a disease or disorder having an etiology associated with excessive cell proliferation with a composition comprising cardiac glycoside, the method comprising:
 a) obtaining a sample of diseased tissue from the subject, the disease having an etiology associated with excessive cell proliferation and the sample comprising one or more isoforms of the cc subunit of Na, K-ATPase;   b) requesting that the ratio of α3 isoform to α1 isoform of the cc subunit of Na, K-ATPase in the sample be determined with a kit of  claim 47 ; and   c) indicating administration to the subject a composition comprising cardiac glycoside if the ratio is ≧0.3.   
     
     
         60 ) The method of  claim 58 , wherein the subject is prescribed and administered a therapeutically relevant dose of composition comprising cardiac glycoside according to a prescribed dosing regimen. 
     
     
         61 ) The method of  claim 60 , wherein the subject is administered the composition comprising cardiac glycoside according to a prescribed dosing regimen comprising repeated administration of one or more therapeutically relevant doses at specified time intervals throughout a specified treatment period. 
     
     
         62 ) The method of  claim 61 , wherein the subject is administered a composition comprising an extract comprising a cardiac glycoside. 
     
     
         63 ) The method of  claim 62 , wherein the extract further comprises one or more other therapeutically effective agents. 
     
     
         64 ) The method of  claim 58 , wherein the composition further comprises one or more other therapeutically effective agents. 
     
     
         65 ) The method of  claim 58 , wherein the subject is administered a hot water extract of a plant or animal source comprising cardiac glycoside. 
     
     
         66 ) The method of  claim 58 , wherein the subject is administered 2 mg to 22.5 mg of cardiac glycoside per day. 
     
     
         67 ) The method of  claim 58 , wherein the subject is administered a supercritical fluid extract of a plant or animal source comprising 0.6 to 4.8 mg of cardiac glycoside. 
     
     
         68 ) The method of  claim 58 , wherein the subject is administered a composition comprising 10 to 500 micrograms of pure cardiac glycoside. 
     
     
         69 ) The kit of  claim 47  wherein the instructions and information comprise instructions to:
 a) determine the ratio of c′3 isoform to α1 isoform of Na, K-ATPase in a sample obtained directly or indirectly from diseased cellular tissue of the subject; and 
 b) determine the probability of at least a partial therapeutic response in the subject were the subject to be treated with a therapeutically relevant dose of cardiac glycoside according to the following table: 
 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                     
                   Probability that there will be at least a 
                 
                     
                   Ratio 
                   partial therapeutic response in the subject 
                 
                     
                     
                 
                     
                   0.3-0.45 +/− 0.05 
                   20-<30% 
                 
                     
                   0.5-0.95 +/− 0.05 
                     30-50% 
                 
                     
                   >/=1 +/− 0.05 
                   >50%  
                 
                     
                   >10 
                   >75%. 
                 
                     
                     
                 
             
                
                
                
                
               
               
                
                
                
                
                
               
            
           
         
       
     
     
         70 ) The kit of  claim 47  comprising instruction to quantify the level of expression of each the α3 subunit isoform of Na, K-ATPase and the α1 subunit isoform of Na, K-ATPase in the in vitro sample or biopsy sample, and calculate the ratio thereof. 
     
     
         71 ) The kit of  claim 70  further comprising instruction to conduct a statistical analysis on data from which the ratio is determined. 
     
     
         72 ) The kit of  claim 47  comprising instruction to lyse or disrupt cells, tissues or biopsy samples; or to fix tissue sections for histopathologic examination from diseased in vivo cellular tissue to form the sample. 
     
     
         73 ) The kit of  claim 72  further comprising instruction to: perform a Western blot assay or immunohistochemical staining assay on the sample to determine the amount and relative expression of α3 subunit isoform of Na, K-ATPase and of the α1 subunit isoform of Na, K-ATPase in the sample; and to calculate the ratio thereof. 
     
     
         74 ) The kit of  claim 47  comprising instruction to: compare the content of α3 subunit isoform of Na, K-ATPase and of α1 subunit isoform of Na, K-ATPase in the sample relative to the content of α3 subunit isoform of Na, K-ATPase and/or of α1 subunit isoform of Na, K-ATPase in a positive control sample and/or a negative control sample. 
     
     
         75 ) The kit of  claim 47  further comprising instruction to identify a subject having a disease or disorder having an etiology associated with excessive cell proliferation. 
     
     
         76 ) The kit of  claim 75  further comprising: obtaining a sample of diseased cells from the subject. 
     
     
         77 ) (canceled) 
     
     
         78 ) (canceled) 
     
     
         79 ) (canceled) 
     
     
         80 ) The kit of  claim 47  comprising instruction to conduct a radiometric or densitometric analysis of a gel in order to determine the content of α3 subunit isoform of Na, K-ATPase and the content of α1 subunit isoform of Na, K-ATPase in the sample; and to determine the ratio of α3 isoform to α1 isoform present in the sample. 
     
     
         81 ) (canceled) 
     
     
         82 ) The kit of  claim 47  comprising instruction to:
 a) provide a sample of mammalian tissue; 
 b) immunochemically stain the α3 isoform and α1 isoforms of the α-subunit of Na, K-ATPase present in the sample; 
 c) determine the content of α3 isoform of Na, K-ATPase and the content of α1 isoform of Na, K-ATPase in the sample; and 
 d) determine the ratio of α3 isoform to α1 isoform present in the sample. 
 
     
     
         83 ) The kit of  claim 47 , wherein the disease or disorder having an etiology associated with excessive cell proliferation is selected from the group consisting of: 1) autoimmune diseases such as antigen-induced arthritis and allergic encephalomyelitis; 2) chronic inflammatory proliferative diseases such as rheumatoid arthritis, systemic-onset juvenile chronic arthritis, osteoporosis, and psoriasis; 3) proliferative diseases of the breast including fibrocystic disease; 4) proliferative diseases of the prostate including benign prostatic hyperplasia; 5) proliferative diseases of the eye including proliferative diabetic retinopathy; 6) vascular proliferative diseases including atherosclerosis and coronary stenosis; 7) cancer; and/or 8) tumor. 
     
     
         84 ) The kit of  claim 47 , wherein the cardiac glycoside is selected from the group consisting of oleandrin, odoroside, neritaloside, ouabain, bufalin, digitoxin, digoxin, cinobufatalin, cinobufagin, and resibufogenin. 
     
     
         85 ) The kit of  claim 47  comprising instructions to:
 obtain a sample of diseased tissue from the subject, the disease having an etiology associated with excessive cell proliferation, and the sample comprising one or more isoforms of the α subunit of Na, K-ATPase; 
 determine the ratio of α3 isoform to α1 isoform of the α subunit of Na, K-ATPase in the sample; and 
 if the ratio is ≧0.3, indicate that the subject should be treated with cardiac glycoside by administration of a composition comprising cardiac glycoside to the subject according to a prescribed dosing regimen, or 
 if the ratio is <0.3, indicate that the subject should not be treated with cardiac glycoside for treatment of the disease or disorder having an etiology associated with excessive cell proliferation. 
 
     
     
         86 ) The kit of  claim 85 , wherein if the ratio is ≧0.5, ≧1, or ≧10, comprising instructions indicating that the subject should be treated with cardiac glycoside by administration of a composition comprising cardiac glycoside to the subject according to a prescribed dosing regimen.