Method for Determining the Stability of Organic Methyleneamines in the Presence of Semicarbazide-Sensitive Amine Oxidase
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, wherein the test agent is a drug candidate and the metabolic stability of the test agent due to SSAO catalyzed metabolism is unknown, 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 measured in step (d) to a control to determine the metabolic stability of the test agent in the presence of cells comprising expressed SSAO.
2 . The method of claim 1 , wherein the cells expressing SSAO are produced by transient or stable transfection of the cells with DNA encoding SSAO.
3 . The method of claim 1 , wherein the cells expressing SSAO are eukaryotic.
4 . The method of claim 1 , 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.
5 . The method of claim 1 , wherein one or more steps are performed by a robotic device.
6 . The method of claim 1 , 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.
7 . The method of claim 1 , wherein the control is a condition in which the test agent is incubated for the predetermined period of time in the absence of cells expressing SSAO or in cell lysates obtained from such cells.
8 . The method of claim 2 , wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 2.
9 . The method of claim 2 , wherein the DNA encodes the amino acid sequence of SEQ ID NO: 1.