Metabolic Stability Study

Liver microsomes are obtained from liver tissue homogenates by differential centrifugation. Liver microsomes are rich in various metabolic enzymes, especially CYP450 enzymes, and are easy to store and use at relatively low cost. Hepatocytes maintain a complete cellular structure with a more complete metabolic enzyme system and coenzyme factors of different clearance pathways. In addition, unlike liver microsomes, hepatocytes also retain cell membrane and membrane transporter proteins. The main reasons for the difference between the results of liver microsomal stability and hepatocyte stability assay are as follows:

1.When a compound has poor membrane permeability or is an efflux transporter substrate, the compound cannot be fully exposed to metabolic enzymes in the hepatocyte system, resulting in a slow metabolic rate of the compound in the hepatocyte.

2.Non-CYP mediated metabolism includes Phase I (e.g., AO, MAO) and Phase II (e.g., UGT). Most of these enzymes either do not exist in liver microsomes or lack the cofactor of UGT in liver microsomes. In contrast, these enzymes are present in hepatocytes. Therefore, compounds primarily metabolized by non-CYP enzymes such as AO or MAO may exhibit certain differences in results between liver microsomal and hepatocyte stability assay.

Other empirical statistical studies have found that the stability of liver microsomes is related to the physicochemical properties, permeability and enzyme phenotype of the compounds, among which it is found that compounds mainly metabolized by CYP3A have a higher intrinsic clearance value in liver microsomal than in hepatocyte stability assay, and there was no significant difference in the metabolism of CYP2D6 substrates between liver microsomes and hepatocyte.

If a compound does not show significant metabolism in metabolic stability studies using liver microsomes and hepatocytes in suspension, with a remaining close to 100% at the longest incubation time point, relay method using hepatocytes in suspension or the plated hepatocytes culture method is more suitable for accurately calculating the clearance value of such compounds.

In recent years, more and more attention has been paid to the study of Non-P450 enzyme-mediated metabolism. The main reason is that researchers introduce chemical groups that are not subject to P450 mediated metabolism, thereby increasing the proportion of involvement from Non-P450 enzymes.

In order to investigate whether the compound is metabolized by Non-P450 enzymes, the appropriate matrix should be selected according to the type of metabolic enzymes in the presence or absence of an inhibitor. Stability experiments can also be performed using recombinant enzymes. The main non-P450 enzymes in phase Ⅰ metabolism are aldehyde oxidase (AO), xanthine oxidase (XO), aldo-keto reductase (AKR), flavin monooxygenase (FMO) and monoamine oxidase (MAO). These five enzymes account for about 5% of the metabolic participation rate among the Top 200 most prescribed drugs from 2005 to 2016.