Methods for detecting lp-pla2 activity and inhibition of lp-pla2 activity

Abstract

This invention relates to methods for determining the activity of Lp-PLA2 in at least one sample from an animal. The invention also relates to methods for determining the inhibition of Lp-PLA2 activity in samples from animals that are administered an Lp-PLA2 inhibitor.

What is claimed is: 
     
         1 . A colorimetric or fluorometric method of determining the percentage of inhibition of Lp-PLA2 activity, the method comprising:
 colorimetrically or fluorometrically detecting Lp-PLA2 activity from a first blood sample that is taken from the subject at a first time and diluted less than 33 times;   colorimetrically or fluorometrically detecting Lp-PLA2 activity from a second blood sample that is taken from the subject at a second time one hour or more after the first time and diluted less than 33 times; and   detecting greater than 30% inhibition of Lp-PLA2 activity between the first and second blood samples by comparing the activity of the Lp-PLA2 between the first and second blood samples, indicating that the subject has taken a drug that inhibits Lp-PLA2.   
     
     
         2 . The method of  claim 1 , further comprising preparing a solution comprising a substrate for Lp-PLA2 having a colorimetric or fluorometric detectable moiety. 
     
     
         3 . The method of  claim 1 , wherein the second blood sample is taken after the subject has been administered a drug that inhibits Lp-PLA2. 
     
     
         4 . The method of  claim 1 , wherein detecting greater than 30% inhibition of Lp-PLA2 activity comprises detecting greater than 90% inhibition of Lp-PLA2 between the first and second blood samples 
     
     
         5 . The method of  claim 1 , wherein detecting greater than 30% inhibition of Lp-PLA2 activity comprises detecting 85-95% inhibition of Lp-PLA2 between the first and second blood samples. 
     
     
         6 . The method of  claim 1 , wherein the first and second blood samples comprise blood plasma or blood serum samples. 
     
     
         7 . The method of  claim 1 , wherein colorimetrically or fluorometrically detecting Lp-PLA2 activity from the first and second blood samples comprises detecting Lp-PLA2 activity within a dynamic range of 100-fold or greater. 
     
     
         8 . A method of clinically monitoring a patient to assess the efficacy of an inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), the method comprising:
 receiving a sample from an animal that has been administered an Lp-PLA2 inhibitor; and   colorimetrically or fluorometrically detecting greater than 30 percent inhibition of Lp-PLA2 from the sample by contacting the sample with a solution comprising a substrate for Lp-PLA2.   
     
     
         9 . The method of  claim 8 , wherein colorimetrically or fluorometrically detecting greater than 30 percent inhibition comprises detecting greater than 70% inhibition. 
     
     
         10 . The method of  claim 8 , wherein colorimetrically or fluorometrically detecting greater than 30 percent inhibition comprises detecting at least 85-75% inhibition. 
     
     
         11 . The method of  claim 8 , wherein colorimetrically or fluorometrically detecting greater than 30 percent inhibition comprises detecting 85-75% inhibition. 
     
     
         12 . The method of  claim 8 , wherein colorimetrically or fluorometrically detecting comprises contacting the sample with a solution so that the sample is diluted less than 33 fold. 
     
     
         13 . The method of  claim 8 , wherein the substrate for Lp-PLA2 includes a colorimetrically or fluorometrically detectable moiety. 
     
     
         14 . The method of  claim 8 , wherein receiving a sample comprises receiving a blood plasma or blood serum sample. 
     
     
         15 . The method of  claim 8 , wherein colorimetrically or fluorometrically detecting comprises detecting inhibition of Lp-PLA2 from a plurality of samples obtained from the animal at more than one time point before and after administration of Lp-PLA2 inhibitor. 
     
     
         16 . The method of  claim 8 , wherein colorimetrically or fluorometrically detecting comprises comparing the activity of Lp-PLA2 from a plurality of samples including at least one sample obtained from the animal prior to the administration of Lp-PLA2 inhibitor. 
     
     
         17 . The method of  claim 8 , wherein the substrate concentration is between about 53 μM to about 1125 μM. 
     
     
         18 . The method of  claim 8 , wherein the substrate concentration is less than 10 times the Km of Lp-PLA2 for the given substrate. 
     
     
         19 . The method of  claim 8 , wherein the animal is human. 
     
     
         20 . A method of determining inhibition of lipoprotein-associated phospholipase A2 (Lp-PLA2) enzyme activity from a sample, the method comprising:
 preparing a solution comprising a substrate for Lp-PLA2;   contacting a sample from an animal that has been administered an Lp-PLA2 inhibitor, wherein the sample is diluted less than a 33 fold by contact with the solution; and   colorimetrically or fluorometrically detecting Lp-PLA2 activity.   
     
     
         21 . The method of  claim 20 , wherein colorimetrically or fluorometrically detecting Lp-PLA2 activity comprises detecting greater than 30 percent inhibition. 
     
     
         22 . The method of  claim 20 , wherein colorimetrically or fluorometrically detecting Lp-PLA2 activity comprises detecting at least 85-95% inhibition. 
     
     
         23 . The method of  claim 20 , wherein contacting the sample from the animal comprises contacting a blood plasma or blood serum sample. 
     
     
         24 . The method of  claim 20 , wherein colorimetrically or fluorometrically detecting comprises detecting activity of Lp-PLA2 from a plurality of samples obtained from the animal at more than one time point after administration of Lp-PLA2 inhibitor. 
     
     
         25 . The method of  claim 20 , wherein colorimetrically or fluorometrically detecting comprises comparing the activity of Lp-PLA2 from a plurality of samples including at least one sample obtained from the animal prior to the administration of Lp-PLA2 inhibitor. 
     
     
         26 . The method of  claim 20 , wherein the substrate concentration is between about 53 μM to about 1125 μM. 
     
     
         27 . The method of  claim 20 , wherein the substrate concentration is less than 10 times the Km of Lp-PLA2 for the given substrate 
     
     
         28 . The method of  claim 20 , wherein the animal is human. 
     
     
         29 . The method of  claim 20 , wherein colorimetrically or fluorometrically detecting Lp-PLA2 activity comprises detecting Lp-PLA2 activity within a dynamic range of close to 100-fold. 
     
     
         30 . A colorimetric or fluorometric method of detecting greater than 30% inhibition of lipoprotein-associated phospholipase A2 (Lp-PLA2) enzyme, the method comprising:
 preparing a solution comprising a substrate for Lp-PLA2;   contacting a sample from an animal that has been administered an Lp-PLA2 inhibitor, wherein the sample is diluted less than 33-fold;   colorimetrically or fluorometrically detecting Lp-PLA2 activity.   
     
     
         31 . The method of  claim 30 , wherein colorimetrically or fluorometrically detecting Lp-PLA2 activity comprises detecting at least 85-95% inhibition. 
     
     
         32 . The method of  claim 30 , wherein contacting the sample from the animal comprises contacting a blood plasma or blood serum sample. 
     
     
         33 . The method of  claim 30 , wherein colorimetrically or fluorometrically detecting comprises detecting inhibition of Lp-PLA2 from a plurality of samples obtained from the animal at more than one time point after administration of the Lp-PLA2 inhibitor. 
     
     
         34 . The method of  claim 30 , wherein colorimetrically or fluorometrically detecting comprises comparing the activity of Lp-PLA2 from a plurality of samples including at least one sample obtained from the animal prior to the administration of Lp-PLA2 inhibitor. 
     
     
         35 . The method of  claim 30 , wherein the substrate concentration is between about 53 μM to about 1125 μM. 
     
     
         36 . The method of  claim 30 , wherein the substrate concentration is less than 10 times the Km of Lp-PLA2 for the given substrate 
     
     
         37 . The method of  claim 30 , wherein the animal is human. 
     
     
         38 . The method of  claim 30 , wherein colorimetrically or fluorometrically detecting Lp-PLA2 activity comprises detecting Lp-PLA2 activity within a dynamic range of 100-fold or greater.