Methods for evaluating gastrointestinal absorption of chemicals

Abstract

Nucleic acids and vectors for interfering with the expression of membrane efflux transport proteins in cells that express such proteins are provided. Also provided are cells and cell lines comprising such nucleic acids and vectors. Methods for screening chemicals and biomolecules for gastrointestinal absorption in animals, and kits for practicing such methods are also provided.

1 . A method for screening compounds for gastrointestinal absorption in an animal comprising inhibiting the expression of at least one membrane efflux transport protein in a cell, contacting the cell with a test compound, measuring transcellular transport of the test compound, and comparing the transcellular transport measurements with reference values for transcellular transport of compounds with no gastrointestinal absorption, low gastrointestinal absorption, moderate gastrointestinal absorption, or high gastrointestinal absorption; said measurements relative to the reference values being predictive of the gastrointestinal absorption of the compound in the body of the animal. 
     
     
         2 . The method of  claim 1 , wherein the membrane efflux transport protein is P-glycoprotein, Multidrug Resistance-Associated Protein 2, or Breast Cancer Resistance Protein. 
     
     
         3 . The method of  claim 1 , wherein inhibiting comprises transforming the cell with a nucleic acid sequence encoding a nucleic acid molecule that interferes with the expression of the at least one membrane efflux transport protein. 
     
     
         4 . A method for screening compounds for gastrointestinal absorption in an animal comprising inhibiting the expression of a first membrane efflux transport protein in a cell, inhibiting the expression of a second membrane efflux transport protein in a second cell, contacting the first and second cells with a test compound, measuring transcellular transport of the test compound in the first and second cells, and comparing the transcellular transport measurements with reference values for transcellular transport of compounds with no gastrointestinal absorption, low gastrointestinal absorption, moderate gastrointestinal absorption, or high gastrointestinal absorption; wherein the measurements indicate the relative contribution of the first and second membrane efflux transport protein to transcellular transport of the test compound and wherein said measurements relative to the reference values are predictive of the gastrointestinal absorption of the compound in the body of the animal. 
     
     
         5 . The method of  claim 4 , wherein the first membrane efflux transport protein is P-glycoprotein, Multidrug Resistance-Associated Protein 2, or Breast Cancer Resistance Protein. 
     
     
         6 . The method of  claim 4 , wherein the second membrane efflux transport protein is P-glycoprotein, Multidrug Resistance-Associated Protein 2, or Breast Cancer Resistance Protein. 
     
     
         7 . The method of  claim 4 , wherein inhibiting the first membrane efflux transport protein comprises transforming the first cell with a nucleic acid sequence encoding a nucleic acid molecule that interferes with the expression of said first membrane efflux transport protein. 
     
     
         8 . The method of  claim 4 , wherein inhibiting the second membrane efflux transport protein comprises transforming the second cell with a nucleic acid sequence encoding a nucleic acid molecule that interferes with the expression of said second membrane efflux transport protein. 
     
     
         9 . The method of  claim 4 , further comprising inhibiting the expression of a third membrane efflux transport protein in a third cell, contacting the first, second, and third cells with a with a test compound, measuring transcellular transport of the test compound in the first, second, and third cells, and comparing the transcellular transport measurements with reference values for transcellular transport of compounds with no gastrointestinal absorption, low gastrointestinal absorption, moderate gastrointestinal absorption, or high gastrointestinal absorption; wherein the measurements indicate the relative contribution of the first, second, and third membrane efflux transport protein to transcellular transport of the test compound and wherein said measurements relative to the reference values are predictive of the gastrointestinal absorption of the compound in the body of the animal. 
     
     
         10 . The method of  claim 9 , wherein the third membrane efflux transport protein is P-glycoprotein, Multidrug Resistance-Associated Protein 2, or Breast Cancer Resistance Protein. 
     
     
         11 . The method of  claim 9 , wherein inhibiting the third membrane efflux transport protein comprises transforming the third cell with a nucleic acid sequence encoding a nucleic acid molecule that interferes with the expression of said third membrane efflux transport protein. 
     
     
         12 . A method for inhibiting the expression of at least one membrane efflux transport protein comprising transforming a cell that expresses at least one membrane efflux transport protein with a lentivirus vector comprising a nucleic acid sequence encoding a nucleic acid molecule that interferes with the expression of at least one membrane efflux transport protein, and expressing the nucleic acid molecule in the cell, wherein expression of the nucleic acid molecule inhibits the expression of at least one membrane efflux transport protein. 
     
     
         13 . The method of  claim 12 , wherein the membrane efflux transport protein is P-glycoprotein, Multidrug Resistance-Associated Protein 2, or Breast Cancer Resistance Protein. 
     
     
         14 . A method for identifying compounds that inhibit the efflux activity of a membrane efflux transport protein comprising inhibiting the expression of a membrane efflux transport protein in a first cell, contacting the first cell with a substrate of the membrane efflux transport protein, contacting a second cell expressing the membrane efflux transport protein with a test compound and a substrate of the membrane efflux transport protein, determining the efflux activity of the membrane efflux transport protein in the first cell, and in the second cell in the presence and absence of the test compound, and comparing the determined efflux activities, wherein a decrease in the efflux activity in the presence of the test compound relative to the efflux activity in the absence of the test compound, and at least partial identity of the efflux activity in the presence of the test compound with the efflux activity in the first cell indicates that the test compound specifically inhibits the membrane efflux transport protein. 
     
     
         15 . The method of  claim 14 , wherein inhibiting comprises transforming the first cell with a nucleic acid sequence encoding a nucleic acid molecule that interferes with the expression of the membrane efflux transport protein.