Methods of classifying, diagnosing, stratifying and treating cancer patients and their tumors

Abstract

The invention provides a variety of reagents for use in the diagnosis and management of cancer, particularly breast cancer. cDNA microarray technology was used to identify genes whose expression profile across a large group of tumor samples correlates with that of cytokeratin 5 and cytokeratin 17, markers for basal cells of the normal mammary lactation gland. The invention demonstrates that tumors that express cytokeratin 5/6 and/or 17 have a poor prognosis relative to tumors overall. The invention provides basal marker genes and their expression products and uses of these genes for diagnosis of cancer and for identification of therapies for cancer. In particular, the invention provides basal marker genes including cadherin3, matrix metalloproteinase 14, and cadherin EGF LAG seven-pass G-type receptor 2. The invention provides antibodies to the polypeptides expressed by these genes and methods of use thereof.

1 . A method of classifying a tumor comprising the steps of: 
 providing a tumor sample;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:1 in the sample; and    classifying the tumor as belonging to a tumor subclass based on the results of the detecting step.    
     
     
         2 . A method of classifying a tumor comprising the steps of: 
 providing a tumor sample;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:2 in the sample; and    classifying the tumor as belonging to a tumor subclass based on the results of the detecting step.    
     
     
         3 . A method of classifying a tumor comprising the steps of: 
 providing a tumor sample;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:3 in the sample; and    classifying the tumor as belonging to a tumor subclass based on the results of the detecting step.    
     
     
         4 . A method of classifying a tumor comprising the steps of: 
 providing a tumor sample;    detecting expression or activity of at least two genes selected from the group consisting of: a gene encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the sample; and    classifying the tumor as belonging to a tumor subclass based on the results of the detecting step.    
     
     
         5 . The method of any of claims  1 ,  2 ,  3 , or  4 , wherein the detecting step comprises detecting the polypeptide or polypeptides.  
     
     
         6 . The method of  claim 5 , wherein the polypeptide is detected by performing immunohistochemical analysis on the sample using an antibody that specifically binds to the polypeptide.  
     
     
         7 . The method of  claim 5 , wherein the polypeptide is detected by performing an ELISA assay using an antibody that specifically binds to the polypeptide.  
     
     
         8 . The method of  claim 5 , wherein the polypeptide is detected using an antibody array comprising an antibody that specifically binds to the polypeptide.  
     
     
         9 . The method of  claim 5 , wherein the detecting step comprises: 
 detecting modification of a substrate by the polypeptide.    
     
     
         10 . The method of any of claims  1 ,  2 ,  3 , or  4 , wherein classifying a tumor comprises: 
 stratifying a subject having the tumor for a clinical trial.    
     
     
         11 . The method of  claim 10 , wherein the tumor is a breast tumor.  
     
     
         12 . The method of any of claims  1 ,  2 ,  3 , or  4 , wherein the tumor is a breast tumor and the tumor subclass is a basal tumor subclass.  
     
     
         13 . The method of  claim 1 , further comprising: 
 providing diagnostic, prognostic, or predictive information based on the classifying step.    
     
     
         14 . The method of  claim 2 , further comprising: 
 providing diagnostic, prognostic, or predictive information based on the classifying step.    
     
     
         15 . The method of  claim 3 , further comprising: 
 providing diagnostic, prognostic, or predictive information based on the classifying step.    
     
     
         16 . The method of  claim 4 , further comprising: 
 providing diagnostic, prognostic, or predictive information based on the classifying step.    
     
     
         17 . The method of  claim 5 , further comprising: 
 providing diagnostic, prognostic, or predictive information based on the classifying step.    
     
     
         18 . The method of  claim 17 , wherein the polypeptide is detected by performing immunohistochemical analysis on the sample using an antibody that specifically binds to the polypeptide.  
     
     
         19 . The method of  claim 17 , wherein the polypeptide is detected by performing an ELISA assay using an antibody that specifically binds to the polypeptide.  
     
     
         20 . The method of  claim 17 , wherein the polypeptide is detected using an antibody array comprising an antibody that specifically binds to the polypeptide.  
     
     
         21 . The method of  claim 17 , wherein the detecting step comprises: 
 detecting modification of a substrate by the polypeptide.    
     
     
         22 . The method of any of claims  13 ,  14 ,  15 , or  16 , wherein the tumor is a breast tumor and the tumor subclass is a basal tumor subclass.  
     
     
         23 . The method of  claim 1 , further comprising: 
 selecting a treatment based on the classifying step.    
     
     
         24 . The method of  claim 2 , further comprising: 
 selecting a treatment based on the classifying step.    
     
     
         25 . The method of  claim 3 , further comprising: 
 selecting a treatment based on the classifying step.    
     
     
         26 . The method of  claim 4 , further comprising: 
 selecting a treatment based on the classifying step.    
     
     
         27 . The method of  claim 5 , further comprising: 
 selecting a treatment based on the classifying step.    
     
     
         28 . The method of  claim 27 , wherein the polypeptide is detected by performing immunohistochemical analysis on the sample using an antibody that specifically binds to the polypeptide.  
     
     
         29 . The method of  claim 27 , wherein the polypeptide is detected by performing an ELISA assay using an antibody that specifically binds to the polypeptide.  
     
     
         30 . The method of  claim 27 , wherein the polypeptide is detected using an antibody array comprising an antibody that specifically binds to the polypeptide.  
     
     
         31 . The method of  claim 27 , wherein the detecting step comprises: 
 detecting modification of a substrate by the polypeptide.    
     
     
         32 . The method of any of claims  23 ,  24 ,  25 , or  26 , wherein the tumor is a breast tumor and the tumor subclass is a basal tumor subclass.  
     
     
         33 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:1 in the sample; and    providing diagnostic, prognostic, or predictive information based on the detecting step.    
     
     
         34 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:2 in the sample; and    providing diagnostic, prognostic, or predictive information based on the detecting step.    
     
     
         35 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:3 in the sample; and    providing diagnostic, prognostic, or predictive information based on the detecting step.    
     
     
         36 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from the subject;    detecting expression or activity of at least two genes selected from the group consisting of: a gene encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the sample; and    providing diagnostic, prognostic, or predictive information based on the detecting step.    
     
     
         37 . The method of any of claims  33 ,  34 ,  35 , or  36 , wherein the detecting step comprises detecting the polypeptide or polypeptides.  
     
     
         38 . The method of  claim 37 , wherein the polypeptide is detected by performing immunohistochemical analysis on the sample using an antibody that specifically binds to the polypeptide.  
     
     
         39 . The method of  claim 37 , wherein the polypeptide is detected by performing an ELISA assay using an antibody that specifically binds to the polypeptide.  
     
     
         40 . The method of  claim 37 , wherein the polypeptide is detected using an antibody array comprising an antibody that specifically binds to the polypeptide.  
     
     
         41 . The method of  claim 37 , wherein the detecting step comprises: 
 detecting modification of a substrate by the polypeptide.    
     
     
         42 . The method of any of claims  33 ,  34 ,  35 , or  36 , wherein the sample is selected from the group consisting of: 
 a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, and a portion of tissue.    
     
     
         43 . The method of any of claims  33 ,  34 ,  35 , or  36 , wherein the sample is a tumor sample.  
     
     
         44 . The method of  claim 43 , wherein the tumor sample is a breast tumor sample.  
     
     
         45 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:1 in the sample; and    stratifying the subject for a clinical trial based on the detecting step.    
     
     
         46 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:2 in the sample; and    stratifying the subject for a clinical trial based on the detecting step.    
     
     
         47 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:3 in the sample; and    stratifying the subject for a clinical trial based on the detecting step.    
     
     
         48 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from the subject;    detecting expression or activity of at least two genes selected from the group consisting of: a gene encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the sample; and    stratifying the subject for a clinical trial based on the detecting step.    
     
     
         49 . The method of any of claims  45 ,  46 ,  47 , or  48 , wherein the detecting step comprises detecting the polypeptide or polypeptides.  
     
     
         50 . The method of  claim 49 , wherein the polypeptide is detected by performing immunohistochemical analysis on the sample using an antibody that specifically binds to the polypeptide.  
     
     
         51 . The method of  claim 49 , wherein the polypeptide is detected by performing an ELISA assay using an antibody that specifically binds to the polypeptide.  
     
     
         52 . The method of  claim 49 , wherein the polypeptide is detected using an antibody array comprising an antibody that specifically binds to the polypeptide.  
     
     
         53 . The method of  claim 49 , wherein the detecting step comprises: 
 detecting modification of a substrate by the polypeptide.    
     
     
         54 . The method of any of claims  45 ,  46 ,  47 , or  48 , wherein the sample is selected from the group consisting of: 
 a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, and a portion of tissue.    
     
     
         55 . The method of any of claims  45 ,  46 ,  47 , or  48 , wherein the sample is a tumor sample.  
     
     
         56 . The method of  claim 55 , wherein the tumor sample is a breast tumor sample.  
     
     
         57 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:1 in the sample; and    selecting a treatment based on the detecting step.    
     
     
         58 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:2 in the sample; and    selecting a treatment based on the detecting step.    
     
     
         59 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from a subject;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:3 in the sample; and    selecting a treatment based on the detecting step.    
     
     
         60 . A method of testing a subject comprising the steps of: 
 providing a sample isolated from the subject;    detecting expression or activity of at least two genes selected from the group consisting of: a gene encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the sample; and    selecting a treatment based on the detecting step.    
     
     
         61 . The method of any of claims  57 ,  58 ,  59 , or  60 , wherein the detecting step comprises detecting the polypeptide or polypeptides.  
     
     
         62 . The method of  claim 61 , wherein the polypeptide is detected by performing immunohistochemical analysis on the sample using an antibody that specifically binds to the polypeptide.  
     
     
         63 . The method of  claim 61 , wherein the polypeptide is detected by performing an ELISA assay using an antibody that specifically binds to the polypeptide.  
     
     
         64 . The method of  claim 61 , wherein the polypeptide is detected using an antibody array comprising an antibody that specifically binds to the polypeptide.  
     
     
         65 . The method of  claim 61 , wherein the detecting step comprises: 
 detecting modification of a substrate by the polypeptide.    
     
     
         66 . The method of any of claims  57 ,  58 ,  59 , or  60 , wherein the sample is selected from the group consisting of: 
 a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, and a portion of tissue.    
     
     
         67 . The method of any of claims  57 ,  58 ,  59 , or  60 , wherein the sample is a tumor sample.  
     
     
         68 . The method of  claim 67 , wherein the tumor sample is a breast tumor sample.  
     
     
         69 . An antibody that specifically binds to an epitope found in a polypeptide whose amino acid sequence the amino acid sequence of SEQ ID NO:1, and wherein the antibody recognizes basal cells in normal mammary lactation glands.  
     
     
         70 . The antibody of  claim 69 , wherein the antibody distinguishes basal cells from luminal cells in normal mammary lactation glands.  
     
     
         71 . The antibody of  claim 69 , wherein the antibody is a monoclonal antibody.  
     
     
         72 . The antibody of  claim 69 , wherein the antibody is a polyclonal antibody.  
     
     
         73 . The antibody of  claim 69 , wherein the antibody recognizes an epitope found in a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.  
     
     
         74 . An antibody that specifically binds to an epitope found in a polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:2, and wherein the antibody recognizes basal cells in normal mammary lactation glands.  
     
     
         75 . The antibody of  claim 73 , wherein the antibody distinguishes basal cells from luminal cells in normal mammary lactation glands.  
     
     
         76 . The antibody of  claim 73 , wherein the antibody is a monoclonal antibody.  
     
     
         77 . The antibody of  claim 73 , wherein the antibody is a polyclonal antibody.  
     
     
         78 . The antibody of  claim 73 , wherein the antibody recognizes an epitope found in a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.  
     
     
         79 . An antibody that specifically binds to an epitope found in a polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:3, and wherein the antibody recognizes basal cells in normal mammary lactation glands.  
     
     
         80 . The antibody of  claim 79 , wherein the antibody distinguishes basal cells from luminal cells in normal mammary lactation glands.  
     
     
         81 . The antibody of  claim 79 , wherein the antibody is a monoclonal antibody.  
     
     
         82 . The antibody of  claim 79 , wherein the antibody is a polyclonal antibody.  
     
     
         83 . The antibody of  claim 79 , wherein the antibody recognizes an epitope found in a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.  
     
     
         84 . A kit for tumor diagnosis comprising: 
 one or more of the antibodies of any of claims  68  through  82 ;    instructions for use of the kit; and    a control slide comprising breast tissue samples for testing reagents in the kit.    
     
     
         85 . A method of testing a compound or a combination of compounds for activity against tumors comprising steps of: 
 obtaining or providing tumor samples taken from subjects who have been treated with the compound or combination of compounds, wherein the tumors fall within a tumor subclass;    comparing the response rate of tumors that fall within the tumor subclass and have been treated with the compound with the overall response rate of tumors that have been treated with the compound or combination of compounds or with the response rate of tumors that do not fall within the subclass and have been treated with the compound or combination of compounds; and    identifying the compound or combination of compounds as having selective activity against tumors in the tumor subclass if the response rate of tumors in the subclass is greater than the overall response rate or the response rate of tumors that do not fall within the subclass.    
     
     
         86 . The method of  claim 85 , wherein the tumors are breast tumors.  
     
     
         87 . The method of  claim 86 , wherein the tumor subclass is a basal tumor subclass.  
     
     
         88 . The method of  claim 86 , wherein the tumors are classified according to the method of any of claims  1 ,  2 ,  3 , or  4 .  
     
     
         89 . The method of  claim 86 , wherein the tumor subclass is a basal tumor subclass and wherein a tumor is identified as belonging to the tumor subclass based on evidence of expression of one or more basal marker genes in the sample.  
     
     
         90 . The method of  claim 89 , wherein evidence of expression comprises presence of a protein encoded by a basal marker gene, and wherein the evidence of expression is obtained using an antibody that binds to the protein.  
     
     
         91 . The method of  claim 90 , wherein the basal marker gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:1.  
     
     
         92 . The method of  claim 90 , wherein the basal marker gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         93 . The method of  claim 90 , wherein the basal marker gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3.  
     
     
         94 . The method of  claim 85 , wherein the samples are present within a tissue array.  
     
     
         95 . A method of testing a compound or a combination of compounds for activity against tumors comprising steps of: 
 treating subjects in need of treatment for tumors with the compound or combination of compounds;    comparing the response rate of tumors that fall within a tumor subclass with the overall response rate of tumors or with the response rate of tumors that do not fall within the subclass; and    identifying the compound or combination of compounds as having selective activity against tumors in the tumor subclass if the response rate of tumors in the subclass is greater than the overall response rate or the response rate of tumors that do not fall within the subclass.    
     
     
         96 . The method of  claim 95 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    determining whether the tumors fall within a tumor subclass; and    stratifying the subjects based on the results of the determining step prior to performing the treating step.    
     
     
         97 . The method of  claim 95 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:1 in the samples; and    stratifying the subjects based on the results of the detecting step prior to performing the the treating step.    
     
     
         98 . The method of  claim 95 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:2 in the samples; and    stratifying the subjects based on the results of the detecting step prior to performing the treating step.    
     
     
         99 . The method of  claim 95 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:3 in the samples; and    stratifying the subjects based on the results of the detecting step prior to performing the treating step.    
     
     
         100 . The method of  claim 95 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    detecting expression or activity of at least two genes, wherein each of the genes encodes a polypeptide whose sequence comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the samples; and    stratifying the subjects based on the results of the detecting step prior to performing the treating step.    
     
     
         101 . A method of testing a compound or a combination of compounds for activity against tumors comprising steps of: 
 treating subjects in need of treatment for tumors with the compound or combination of compounds or with an alternate compound, wherein the tumors fall within a tumor subclass;    comparing the response rate of tumors treated with the compound or combination of compounds with the response rate of tumors treated with the alternate compound; and    identifying the compound or combination of compounds as having superior activity against tumors in the tumor subclass, as compared with the alternate compound, if the response rate of tumors treated with the compound or combination of compounds is greater than the response rate of tumors treated with the alternate compound.    
     
     
         102 . The method of  claim 101 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    determining whether the tumors fall within a tumor subclass; and    stratifying the subjects based on the results of the determining step prior to performing the treating step.    
     
     
         103 . The method of  claim 101 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:1 in the samples; and    stratifying the subjects based on the results of the detecting step prior to performing the treating step.    
     
     
         104 . The method of  claim 101 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:2 in the samples; and    stratifying the subjects based on the results of the detecting step prior to performing the treating step.    
     
     
         105 . The method of  claim 101 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:3 in the samples; and    stratifying the subjects based on the results of the detecting step prior to performing the treating step.    
     
     
         106 . The method of  claim 101 , further comprising the steps of: 
 providing tumor samples from subjects in need of treatment for tumors;    detecting expression or activity of at least two genes, wherein each of the genes encodes a polypeptide whose sequence comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the samples; and    stratifying the subjects based on the results of the detecting step prior to performing the treating step.    
     
     
         107 . The method of any of claims  101 ,  102 ,  103 ,  104 ,  105 , or  106 , wherein the alternate compound is a compound approved by the U.S. Food and Drug administration for treatment of tumors.  
     
     
         108 . A method of treating a subject comprising steps of: 
 identifying a subject as having a tumor in a basal tumor subclass; and    administering a compound identified according to the method of any of claims  85 ,  86 ,  87 , or  90  to the subject.    
     
     
         109 . A method of treating a subject comprising steps of: 
 identifying a subject as having a tumor in a basal tumor subclass; and    administering a compound identified according to the method of any of claims  95 ,  96 ,  97 ,  98 ,  99 , or  100  to the subject.    
     
     
         110 . A method of treating a subject comprising steps of: 
 identifying a subject as having a tumor in a basal tumor subclass; and    administering a compound identified according to the method of any of claims  101 ,  102 ,  103 ,  104 ,  105 , or  106  to the subject.    
     
     
         111 . A method of treating a subject comprising steps of: 
 providing a subject in need of treatment for cancer;    administering to the subject an antibody that specifically binds to a polypeptide having an amino acid sequence comprising the sequence of SEQ ID NO:1.    
     
     
         112 . A method of treating a subject comprising steps of: 
 providing a subject in need of treatment for a tumor;    administering to the subject an antibody that specifically binds to a polypeptide having an amino acid sequence comprising the sequence of SEQ ID NO:2.    
     
     
         113 . A method of treating a subject comprising steps of: 
 providing a subject in need of treatment for a tumor;    administering to the subject an antibody that specifically binds to a polypeptide having an amino acid sequence comprising the sequence of SEQ ID NO:3.    
     
     
         114 . The method of any of claims  111 ,  112 , or  113 , wherein the tumor is a breast tumor, and wherein the method further comprises the step of: 
 identifying the tumor as belonging to a basal tumor subclass.    
     
     
         115 . The method of any of claims  111 ,  112 , or  113 , wherein the antibody is conjugated with a toxic molecule.  
     
     
         116 . A method of treating a subject comprising steps of: 
 providing a subject in need of treatment for cancer;    administering to the subject a compound that activates or inhibits a gene that encodes an amino acid having a sequence comprising the sequence of SEQ ID NO:1, or that activates or inhibits an expression product of the gene.    
     
     
         117 . A method of treating a subject comprising steps of: 
 providing a subject in need of treatment for a tumor;    administering to the subject a compound that activates or inhibits a gene that encodes an amino acid having a sequence comprising the sequence of SEQ ID NO:2, or that activates or inhibits an expression product of the gene.    
     
     
         118 . A method of treating a subject comprising steps of: 
 providing a subject in need of treatment for a tumor;    administering to the subject a compound that activates or inhibits a gene that encodes an amino acid having a sequence comprising the sequence of SEQ ID NO:3, or that activates or inhibits an expression product of the gene.    
     
     
         119 . A composition comprising: 
 two or more compounds identified according to the method of any of claims  85 ,  95 , or  101 .    
     
     
         120 . A pharmaceutical composition comprising: 
 the composition of  claim 119;  and    a pharmaceutically acceptable carrier.    
     
     
         121 . A composition comprising: 
 a compound identified according to the method of any of claims  85 ,  95 , or  101 ;    a second compound, wherein the second compound is approved by the U.S. Food and Drug administration for the treatment of cancer or has shown potential efficacy against cancer in pre-clinical studies.    
     
     
         122 . A pharmaceutical composition comprising: 
 the composition of  claim 121 , and    a pharmaceutically acceptable carrier.