Method for high throughput determination of in vitro metabolism stability of compound, and applications thereof

Abstract

The present invention provides a method for high throughput determination of the in vitro metabolism stability of a compound, and applications thereof. According to the present invention, the high-throughput problem is solved by using the 96-well plate; the reaction is initiated by adding NADPH in the reverse order while the termination reagent is added to terminate the reaction at the same time so as to solve the acetonitrile volatilization problem; and the control group with no NADPH addition is designed, such that the recovery rate can be calculated so as to find and screen the compounds independent of NADPH metabolism.

Description

technical field [0001] The invention belongs to the field of pharmacokinetic research, relates to a measurement method and its application, in particular to a high-throughput method for measuring the in vitro metabolic stability of a compound and its application. Background technique [0002] There are two types of high-throughput methods for determining the metabolic stability of compounds in vitro. One is to operate in centrifuge tubes, which cannot achieve high throughput and cannot determine the recovery rate; the other is to operate in 96-well plates. High throughput was achieved, but recovery could not be determined. [0003] In the existing method, after mixing microsomes, compounds, and NADPH, the reaction is terminated sequentially according to the time points: when using a centrifuge tube, the mixture is taken out from the reaction system and added to acetonitrile to terminate at each time point. The disadvantage is that it is cumbersome and cannot be simultaneousl...

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Problems solved by technology

[0002] There are two types of high-throughput methods for determining the metabolic stability of compounds in vitro. One is to operate in centrifuge tubes, which cannot achieve high throughput and cannot determine the recovery rate; the other is to operate in 96-well plates. High throughput was achieved, but recovery could not be determined

[0003] In the existing method, after mixing microsomes, compounds, and NADPH, the reaction is terminated sequentially according to the time points: when using a centrifuge tube, the mixture is taken out from the reaction system and added to acetonitrile to terminate at each time point. The disadvantage is that it is cumbersome and cannot be simultaneously Do a lot of compounds, and if the mixing is not sufficient, the mixture taken out may be inconsistent, which will affect the measurement results, and there is no control without NADPH, so the recovery rate cannot be calculated; the existing 96-well plate method is also at every time point, After terminating the reaction with acetonitrile, continue to incubate, because other wells are still reacting, and acetonitrile is a volatile solvent. Wait until the later time point to terminate. There is no control without NADPH, the recovery rate cannot be calculated

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