Cellular-based method for determining the biological activity of defibrotide

Abstract

The present invention relates to cell-based methods for determining the biological activity of defibrotide. In particular, the invention provides a method for assessing the potency of defibrotide by assessing the viability of mammalian cells in the presence of at least one cytotoxic agent and one or more concentrations of defibrotide. Such methods are particularly useful for standardizing pharmaceutical compositions comprising defibrotide.

1 - 15 . (canceled) 
     
     
         16 . A pharmaceutical composition comprising defibrotide at a standardized potency ratio between 0.9 and 1.2 compared with a reference batch of defibrotide, wherein at least 10 μg/mL of the defibrotide protects epithelial cells from damage in vitro, and wherein the potency of the defibrotide is assessed by a method comprising:
 growing mammalian cells in culture; 
 incubating the cells with a solution containing at least one cytotoxic agent and at least one concentration of defibrotide from a sample batch; 
 measuring the viability of the cells after an incubation period; 
 comparing the cell viability to the cell viability for a reference batch of defibrotide; and 
 calculating the potency of the sample batch of defibrotide based on the comparison. 
 
     
     
         17 . The pharmaceutical composition of  claim 16 , wherein the cells are incubated with the cytotoxic agent and at least four different concentrations of defibrotide from the sample batch, and wherein the cell viability is determined for each of the concentrations to create a dose-response curve. 
     
     
         18 . The pharmaceutical composition of  claim 16 , wherein the cell viability measured for the sample batch of defibrotide is compared to a calibration curve obtained from cell viability measurements with the reference batch of defibrotide. 
     
     
         19 . The pharmaceutical composition of  claim 16 , wherein the mammalian cells are human endothelial cells, human epithelial cells, human liver sinusoidal endothelial cells, or human microvascular endothelial cells. 
     
     
         20 . The pharmaceutical composition of  claim 16 , wherein the mammalian cells are present at a density of about 5×10 4  cells/ml to about 5×10 5  cells/ml. 
     
     
         21 . The pharmaceutical composition of  claim 16 , wherein the cytotoxic agent is fludarabine, 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-Ara-A), or doxorubicin. 
     
     
         22 . The pharmaceutical composition of  claim 21 , wherein fludarabine or F-Ara-A is present in the solution at a concentration of about 10 μg/ml to about 50 μg/ml or wherein doxorubicin is present in the solution at a concentration of about 0.1 μg/ml to about 10 μg/ml. 
     
     
         23 . The pharmaceutical composition of  claim 16 , wherein the at least one concentration of defibrotide from the sample batch is in the range of about 1 μg/ml to about 100 μg/ml. 
     
     
         24 . The pharmaceutical composition of  claim 16 , wherein the incubation period is at least 24 hours, at least 48 hours, or at least 72 hours. 
     
     
         25 . The pharmaceutical composition of  claim 16 , wherein measuring cell viability comprises performing a colorimetric assay based on the reduction of tetrazolium dyes. 
     
     
         26 . The pharmaceutical composition of  claim 25 , wherein the tetrazolium dye is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium. 
     
     
         27 . The pharmaceutical composition of  claim 16 , wherein measuring cell viability comprises measuring the absorbance of the solution following incubation with the cells. 
     
     
         28 . The pharmaceutical composition of  claim 16 , wherein the sample batch of defibrotide is extracted from bovine tissue or porcine tissue. 
     
     
         29 . A pharmaceutical composition comprising defibrotide at a standardized potency ratio between 0.9 and 1.2 compared with a reference batch of defibrotide, wherein at least 10 μg/mL of the defibrotide protects epithelial cells from damage in vitro, and wherein the potency of the defibrotide is assessed by a method comprising:
 growing mammalian cells in culture in a multi-well microtiter plate; 
 incubating the cells with a solution containing at least one cytotoxic agent and at least four different concentrations of defibrotide from a sample batch; 
 collecting dose response data points simultaneously; 
 measuring the viability of the cells after an incubation period; 
 comparing the cell viability to the cell viability for a reference batch of defibrotide; and 
 calculating the potency of the sample batch of defibrotide based on the comparison. 
 
     
     
         30 . The pharmaceutical composition of  claim 29 , wherein the cells are incubated with a solution containing defibrotide at a concentration between 1.25 to 80 μg/mL. 
     
     
         31 . The pharmaceutical composition of  claim 29 , wherein the cell viability is determined for each of the concentrations to create a dose-response curve. 
     
     
         32 . The pharmaceutical composition of  claim 29 , wherein the cytotoxic agent fludarabine or F-Ara-A is present in the solution at a concentration of about 10 μg/ml. 
     
     
         33 . The pharmaceutical composition of  claim 29 , wherein the multi-well microtiter plate is a 96-well plate. 
     
     
         34 . The pharmaceutical composition of  claim 29 , wherein at least 3 replicates of incubating the cells with a solution containing at least one cytotoxic agent and at least four different concentrations of defibrotide from the sample batch is performed.