Toxicity screening methods

Abstract

Methods of evaluating an anti-tumor compound based on in vitro assays are disclosed. The methods may determine a potency/efficacy of the anti-tumor compound as well as a toxicity of the anti-tumor compound to non-cancerous cells.

1 . A method of determining a level of toxicity in normal tissue for an anti-tumor compound, the method comprising the steps of:
 (a) culturing at least one first cell type in the presence of a plurality of concentrations of an anti-tumor compound, wherein the first cell type is a proliferating cell derived from a tumor;   (b) culturing at least one second cell type in the presence of a plurality of concentrations of the anti-tumor compound, wherein the second cell type is a cell derived from a normal tissue of the same species and tissue as the first cell type;   (c) culturing at least one third cell type in the presence of a plurality of concentrations of the anti-tumor compound, wherein the third cell type is derived from a normal tissue of the same species but different tissue as the first and second cell types;   (d) measuring at least one indicator of cellular replication, at least one indicator of mitochondrial function, and at least one indicator of cell viability at four or more concentrations of the anti-tumor compound for each of the at least three cell types;   (e) determining a potency/efficacy of the anti-tumor compound based on the measurements of the at least one indicator of cellular replication in the first cell type;   (f) determining a specificity of the anti-tumor compound by comparing at least one of the measurements from (d) for at least one of the indicator(s) of cellular replication, mitochondrial function, and cell viability for the first cell type to at least one of the measurements from (d) for at least one of the indicator(s) of cellular replication, mitochondrial function, and cell viability for the second cell type; and   (g) comparing the results obtained for at least one of the indicator(s) of cellular replication, mitochondrial function, and cell viability for the at least one third cell type to the results obtained for the same indicator(s) for the first and second cell types.   
     
     
         2 . The method of  claim 1 , further comprising determining a concentration of the anti-tumor compound that produces a half maximal toxic effect (TC 50 ) for each of the indicator of cellular replication, indicator of mitochondrial function, and indicator of cell viability for each of the first and second cell types. 
     
     
         3 . The method of  claim 2 , further comprising comparing the TC 50  for the indicator of cellular replication in the first cell type to the TC 50  for at least one of the indicators of mitochondrial function and cell viability for the second cell type. 
     
     
         4 . The method of  claim 1 , wherein the plurality of concentrations of the anti-tumor compound are selected from a concentration range from 0 micromolar and to about 300 micromolar. 
     
     
         5 . The method of  claim 1 , further comprising plotting the measurements for each cell health indicator on a graph as a function of concentration for each cell health indicators of the anti-tumor compound. 
     
     
         6 . The method of  claim 1 , wherein the measurements of each of the cell health indicators are expressed relative to a control measurement as a function of concentration of the anti-tumor compound. 
     
     
         7 . The method of  claim 6 , wherein the measurements of all of the cell health indicators are plotted on a single graph. 
     
     
         8 . The method of  claim 1 , further comprising measuring at least one additional indicator of cell health selected from the group consisting of 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 , further comprising the steps of:
 determining a relative abundance of the anti-tumor compound's intended target in each of the at least three cell types; and   normalizing the toxicity response to the target in each cell type.   
     
     
         10 - 20 . (canceled)