Method for determining the stability of organic methyleneamines in the presence of semicarbazide-sensitive amine oxidase

Abstract

The present invention provides methods for determining the stability of methyleneamine, methyleneamine-like compounds or compounds containing an methyleneamine moiety in the presence of semicarbazide-sensitive amine oxidase (SSAO) or a biological sample containing SSAO activity. The disclosed methods may be configured in an assay format for high throughput screening applications.

1 . A method of identifying metabolic stability of a test agent due to semicarbazide-sensitive amine oxidase (SSAO) catalyzed metabolism, the method comprising:
 a) culturing cells expressing SSAO;   b) adding a test agent to the cells;   c) incubating the test agent with the cells for a predetermined period of time;   d) measuring the amount of the test agent remaining in the presence of the cells comprising expressed SSAO at the predetermined period of time; and   e) comparing the amount of the test agent at the predetermined period of time to the amount of test agent in a control to determine a value   
       wherein the value identifies the metabolic stability of the test agent in the presence of cells comprising expressed SSAO. 
     
     
         2 . A method of identifying metabolic stability of a radiolabeled test agent due to semicarbazide-sensitive amine oxidase (SSAO) catalyzed metabolism, the method comprising:
 a) culturing cells expressing SSAO;   b) adding a radiolabeled test agent to the cells;   c) incubating the radiolabeled test agent with the cells for a predetermined period of time; and   d) measuring the amount of the radiolabeled test agent remaining in the presence of the cells comprising expressed SSAO at the predetermined period of time;   
       wherein the percentage of radiolabeled test agent that has not been metabolized identifies the metabolic stability of the radiolabeled test agent in the presence of the cells comprising expressed SSAO. 
     
     
         3 . The method of  claim 1  or  claim 2  wherein the cells expressing SSAO are produced by transient or stable transfection of the cells with DNA encoding SSAO. 
     
     
         4 . The method of  claim 1  or  claim 2  wherein the cells expressing SSAO are prokaryotic. 
     
     
         5 . The method of  claim 1  or  claim 2  wherein the cells expressing SSAO are eukaryotic. 
     
     
         6 . A method of identifying metabolic stability of a test agent due to SSAO catalyzed metabolism in a biological sample, the method comprising:
 a) obtaining a biological sample comprising SSAO;   b) adding a test agent to the biological sample;   c) incubating the test agent with the biological sample for a predetermined period of time;   d) measuring the amount of the test agent at the predetermined period of time; and   e) comparing the amount of the test agent at the predetermined period of time to the amount of test agent in a control to determine a value   
       wherein the value identifies the metabolic stability of the test agent in the presence of a biological sample comprising SSAO. 
     
     
         7 . A method of identifying metabolic stability of a radiolabeled test agent due to SSAO catalyzed metabolism in a biological sample, the method comprising:
 a) obtaining a biological sample comprising SSAO;   b) adding a radiolabeled test agent to the biological sample;   c) incubating the radiolabeld test agent with the biological sample for a predetermined period of time; and   d) measuring the amount of the radiolabeled test agent at the predetermined period of time   
       wherein the percentage of radiolabeled test agent that has not been metabolized identifies the metabolic stability of the test agent in the presence of a biological sample comprising SSAO. 
     
     
         8 . The method of  claim 6  or  claim 7  wherein the biological sample is selected from the group consisting of blood, plasma, serum, artery, umbilical cord, liver, ileum, and lung. 
     
     
         9 . The method of  claim 8  wherein the biological sample is from a human subject. 
     
     
         10 . The method of  claim 8  wherein the biological sample is from a non-human subject. 
     
     
         11 . The method of  claim 9  or  claim 10  wherein the human or non-human subject is manipulated prior to obtaining the biological sample. 
     
     
         12 . The method of  claim 11  wherein the manipulation of the human or non-human subject is selected from the group consisting of treatment with one or more drugs, induction of one or more aspects of a disease state, surgical manipulation and genetic alteration. 
     
     
         13 . The method of  claim 1  or  claim 6  wherein the amount of test agent is determined by a technique selected from the group consisting of mass spectrometry, high-pressure liquid chromatography, liquid chromatography/mass spectrometry, liquid chromatography/mass spectrometry/mass spectrometry and liquid chromatography/radiomatic detection. 
     
     
         14 . The method of  claim 1  or  claim 6  wherein one or more steps are performed by a robotic device. 
     
     
         15 . The method of  claim 1  or  claim 6  wherein the metabolic stability of the test agent is determined by measuring one or more metabolites of the test agent in addition to or in place of measuring the test agent. 
     
     
         16 . A method of determining the metabolic stability profile of a test agent, the method comprising:
 a) obtaining the value of metabolic stability of the test agent incubated in the presence of the cell comprising expressed SSAO using the method of  claim 1 ;   b) obtaining a biological sample comprising SSAO;   c) adding a test agent to the biological sample;   d) incubating the test agent with the biological sample for a predetermined period of time;   e) measuring the amount of the test agent at the predetermined period of time; and   f) comparing the amount of the test agent at the predetermined period of time to the amount of test agent in a control to determine a value that identifies the metabolic stability of the test agent in the presence of a biological sample comprising SSAO   
       wherein the value of metabolic stability of the test agent in the presence of the cell comprising expressed SSAO is compared to the value of metabolic stability of the test agent in the presence of a biological sample comprising SSAO providing the metabolic stability profile of the test agent. 
     
     
         17 . A method of determining the metabolic stability profile of a radiolabeled test agent, the method comprising:
 a) obtaining the value of metabolic stability of the radiolabeled test agent incubated in the presence of the cell lysate comprising expressed SSAO using the method of  claim 2 ;   b) obtaining a biological sample comprising SSAO;   c) adding a radiolabeled test agent to the biological sample;   d) incubating the radiolabeled test agent with the biological sample for a predetermined period of time;   e) measuring the amount of the radiolabeled test agent at the predetermined period of time; and   f) identifying the metabolic stability of the radiolabeled test agent by the percentage of radiolabeled test agent that has not been metabolized at the predetermined period of time   
       wherein the value of metabolic stability of the radiolabeled test agent in the presence of cell lysate comprising expressed SSAO is compared to the value of metabolic stability of the radiolabeled test agent in the presence of a biological sample comprising SSAO providing the metabolic stability profile of the test agent.