Toxicity screening methods

Abstract

The present invention provides methods of determining a level of toxicity of a given compound based on in vitro assays. The present invention provides particular methods of determining organ-specific toxicity and species-specific toxicity of a given compound based on in vitro assays. In addition, the present invention provides methods of determining a level of toxicity in normal tissue for an anti-tumor compound. The methods include providing at least one cell type and culturing the cell type in the presence of at least one concentration of the chemical compound, measuring at least one indicator of cell health at the at least one concentration of compound for the at least one cell type, and performing a concentration response analysis, from which a toxic concentration can be determined.

1 . A method of determining a level of toxicity of a chemical compound comprising the steps of:
 (a) culturing cells in the presence of a plurality of concentrations of said chemical compound;   (b) measuring a first indicator of cell health at four or more concentrations of said chemical compound at two or more time points;   (c) measuring a second indicator of cell health at four or more concentrations of said chemical compound at two or more time points;   (d) measuring a third indicator of cell health at four or more concentrations of said chemical compound at two or more time points;   (e) determining a level of toxicity of said chemical compound from the measurements taken in steps (b), (c) and (d) by:
 (1) performing a concentration response analysis for each indicator of cell health from the measurements taken in steps (b), (c) and (d); 
 (2) identifying, from the concentration response analyses, the highest concentration of said chemical compound at which no measurable toxic effect was observed for each measured indicator of cell health; and 
 (3) selecting, as the toxic concentration (C tox ), a concentration less than or equal to the highest concentration of said chemical compound at which no measurable toxic effect was observed for all measured indicators of cell health, as a level of toxicity for the chemical compound. 
   
     
     
         2 . The method of  claim 1 , wherein each said first, second and third indicators is independently selected from the group consisting of indicators of cellular replication, indicators of mitochondrial function, indicators of intracellular energy balance, indicators of cell membrane integrity, indicators of cell mortality, indicators of oxidative stress, indicators of metabolic stability, indicators of metabolic activation, indicators of enzyme induction, indicators of enzyme inhibition, and indicators of interaction with cell membrane transporters. 
     
     
         3 . The method of  claim 1 , further comprising determining a concentration of said compound that produces a half maximal toxic effect (TC 50 ) for each of said indicators of cell health. 
     
     
         4 . The method of  claim 1 , wherein said plurality of concentrations are selected from a concentration range from 0 micromolar and to about 300 micromolar. 
     
     
         5 . The method of  claim 1 , wherein step (1) comprises plotting the measurements for each said cell health indicators on a graph as a function of concentration for each said cell health indicators of the chemical compound, and wherein the measurements of each of said cell health indicators are expressed relative to a control measurement as a function of concentration of the chemical compound, and wherein the measurements of all of said cell health indicators are plotted on a single graph. 
     
     
         6 . The method of  claim 1 , wherein at least one of said cell health indicators is measured from at least one of the supernatant and cellular components of said cell culture. 
     
     
         7 . The method of  claim 1 , further comprising measuring a fourth cell health indicator selected from the group consisting of indicators of cellular replication, indicators of mitochondrial function, indicators of intracellular energy balance, indicators of cell membrane integrity, indicators of cell mortality, indicators of oxidative stress, indicators of metabolic stability, indicators of metabolic activation, indicators of enzyme induction, indicators of enzyme inhibition, and indicators of interaction with cell membrane transporters. 
     
     
         8 . The method of  claim 1 , further comprising measuring a fifth cell health indicator selected from the group consisting of indicators of cellular replication, indicators of mitochondrial function, indicators of intracellular energy balance, indicators of cell membrane integrity, indicators of cell mortality, indicators of oxidative stress, indicators of metabolic stability, indicators of metabolic activation, indicators of enzyme induction, indicators of enzyme inhibition, and indicators of interaction with cell membrane transporters. 
     
     
         9 . The method of  claim 1 , wherein said cells are primary cells. 
     
     
         10 . The method of  claim 1 , wherein said cells are of mammalian origin and are selected from the group consisting of liver cells, kidney cells, brain cells, fibroblast cells, nerve cells, 3-kin cells, lung cells, spleen cells, endometrial cells, cardiac cells, stomach cells, breast cells, stem cells hematopoietic cells, and cell lines derived from any of these cells. 
     
     
         11 . The method of  claim 1 , wherein said cells are from a mammalian cell line. 
     
     
         12 . The method of  claim 11 , wherein said cells are liver cell line cells selected from the group consisting of human liver cell line cells and rodent liver cell line cells. 
     
     
         13 . The method of  claim 1 , wherein said chemical compound is selected from the group consisting of an antimicrobial agent, an anti-tumor agent, an immunomodulator, a neurotransmitter, an agent for treatment or prevention of a central nervous system disease or disorder or cardiovascular disease or disorder; and an anti-inflammatory agent. 
     
     
         14 . A method of determining a level of toxicity of a chemical compound comprising the steps of:
 (a) culturing cells in the presence of a plurality of concentrations of said chemical compound;   (b) measuring a first indicator of cell health at four or more concentrations of said chemical compound, wherein the first indicator of cell health is selected from the group consisting of indicators of oxidative stress, indicators of metabolic stability, indicators of metabolic activation, indicators of enzyme induction, indicators of enzyme inhibition, and indicators of interaction with cell membrane transporters;   (c) measuring a second indicator of cell health at four or more concentrations of said chemical compound;   (d) measuring a third indicator of cell health at four or more concentrations of said chemical compound;   (e) determining a level of toxicity of said chemical compound from the measurements taken in steps (b), (c) and (d) by:
 (1) performing a concentration response analysis for each indicator of cell health from the measurements taken in steps (b), (c) and (d); 
 (2) identifying, from the concentration response analyses, the highest concentration of said chemical compound at which no measurable toxic effect was observed for each measured indicator of cell health; and 
 (3) selecting, as the toxic concentration (C tox ), a concentration less than or equal to the highest concentration of said chemical compound at which no measurable toxic effect was observed for all measured indicators of cell health, as a level of toxicity for the chemical compound. 
   
     
     
         15 . The method of  claim 14 , wherein each of the first, second and third indicators of cell health are measured at four or more concentrations of said chemical compound at two or more time points. 
     
     
         16 . The method of  claim 14 , wherein each said second and third indicators is independently selected from the group consisting of indicators of cellular replication, indicators of mitochondrial function, indicators of intracellular energy balance, indicators of cell membrane integrity, indicators of cell mortality, indicators of oxidative stress, indicators of metabolic stability, indicators of metabolic activation, indicators of enzyme induction, indicators of enzyme inhibition, and indicators of interaction with cell membrane transporters. 
     
     
         17 . The method of  claim 14 , further comprising determining a concentration of said compound that produces a half maximal toxic effect (TC 50 ) for each of said indicators of cell health. 
     
     
         18 . The method of  claim 14 , wherein said plurality of concentrations are selected from a concentration range from 0 micromolar and to about 300 micromolar. 
     
     
         19 . The method of  claim 14 , wherein step (1) comprises plotting the measurements for each said cell health indicators on a graph as a function of concentration for each said cell health indicators of the chemical compound, and wherein the measurements of each of said cell health indicators are expressed relative to a control measurement as a function of concentration of the chemical compound, and wherein the measurements of all of said cell health indicators are plotted on a single graph. 
     
     
         20 . The method of  claim 14 , wherein at least one of said cell health indicators is measured from at least one of the supernatant and cellular components of said cell culture. 
     
     
         21 . The method of  claim 14 , wherein said cells are primary cells. 
     
     
         22 . The method of  claim 14 , wherein said cells are of mammalian origin and are selected from the group consisting of liver cells, kidney cells, brain cells, fibroblast cells, nerve cells, skin cells, lung cells, spleen cells, endometrial cells, cardiac cells, stomach cells, breast cells, stem cells hematopoietic cells, and cell lines derived from any of these cells. 
     
     
         23 . The method of  claim 14 , wherein said cells are from a mammalian cell line. 
     
     
         24 . The method of  claim 23 , wherein said cells are liver cell line cells selected from the group consisting of human liver cell line cells and rodent liver cell line cells. 
     
     
         25 . The method of  claim 14 , wherein said chemical compound is selected from the group consisting of an antimicrobial agent, an anti-tumor agent, an immunomodulator, a neurotransmitter, an agent for treatment or prevention of a central nervous system disease or disorder or cardiovascular disease or disorder; and an anti-inflammatory agent. 
     
     
         26 . A method of measuring metabolic activation of a chemical compound, comprising the steps of:
 (a) culturing microsomes in the presence of at least one concentration of the chemical compound and a known concentration of reduced glutathione (GSH) in the presence and absence of an inhibitor of cytochrome P450;   (b) culturing microsomes in the presence of at least one concentration of the chemical compound and a known concentration of GSH and in the presence of an inhibitor of cytochrome P450;   (c) measuring an amount of GSH remaining after culturing (a) for a specified amount of time;   (d) measuring an amount of GSH remaining after culturing (b) for a specified amount of time;   (e) determining that the decrease in the amount of GSH in (c) was caused by a cytochrome P450-mediated increase in electrophilic intermediates if (d) is substantially equal to the known concentration of GSH added in (b).   
     
     
         27 . A method of determining a level of toxicity in normal tissue for an anti-tumor compound, comprising the steps of:
 (a) providing at least one first cell type, wherein the first cell type is a proliferating cell derived from a tumor;   (b) providing at least one second cell type, wherein the second cell type is a proliferating cell derived from a normal tissue;   (c) providing at least one third cell type, wherein the third cell type is a non-proliferating cell derived from a normal tissue, and wherein the three cell types are from the same species of mammal;   (d) culturing each of the three cell types in the presence of a plurality of concentrations of said anti-tumor compound;   (e) measuring two or more indicators of cell health at four or more concentrations of said anti-tumor compound for each of the at least three cell types;   (f) determining a level of toxicity of said anti-tumor compound in normal cells from (b) and (c) from the measurements taken in step (e) by:
 (1) performing a concentration response analysis for each of the two or more indicators of cell health from the measurements taken in step (e) for each of the three cell types; 
 (2) identifying, from the concentration response analyses, the highest concentration of said anti-tumor compound at which no measurable toxic effect was observed for each of the two or more indicators of cell health for the second and third cell types; and 
 (3) selecting, as the toxic concentration (C tox ), a concentration less than or equal to the highest concentration of said anti-tumor compound at which no measurable toxic effect was observed for all measured indicators of cell health for the second and third cell types, as a level of toxicity to normal cells for the anti-tumor compound. 
   
     
     
         28 . The method of  claim 27 , wherein each of the two or more indicators is independently selected from the group consisting of indicators of cellular replication, indicators of mitochondrial function, indicators of intracellular energy balance, indicators of cell membrane integrity, indicators of cell mortality, indicators of oxidative stress, indicators of metabolic stability, indicators of metabolic activation, indicators of enzyme induction, indicators of enzyme inhibition, and indicators of interaction with cell membrane transporters. 
     
     
         29 . The method of  claim 27 , further comprising determining a concentration of said compound that produces a half maximal toxic effect (TC 50 ) for each of said indicators of cell health. 
     
     
         30 . The method of  claim 27 , wherein said plurality of concentrations are selected from a concentration range from 0 micromolar and to about 300 micromolar. 
     
     
         31 . The method of  claim 27 , wherein step (1) comprises plotting the measurements for each said cell health indicator for each cell type on a graph as a function of concentration for each said cell health indicator of the chemical compound, and wherein the measurements of each of said cell health indicators are expressed relative to a control measurement as a function of concentration of the chemical compound, and wherein the measurements of all of said cell health indicators for each cell type are plotted on a single graph. 
     
     
         32 . The method of  claim 27 , further comprising the steps of:
 (g) culturing each of the three cell types in the presence of at least one concentration of said anti-tumor compound;   (h) measuring a level of expression of a target molecule to which the chemical compound is directed for each of the three cell types in the presence of the anti-tumor compound; and   (i) determining that the anti-tumor compound is specific for the tumor cells if the level of expression of the target molecule in the first cell type is substantially lower than the level of expression of the target molecule in at least one of the second and third cell types.