Background

Drug metabolism includes a wide range of biochemical processes, which finally lead to either drug inactivation or drug transformation, which facilitates the elimination of xenobiotics from the human body.

Drugs can be metabolized in different organs, such as intestines, kidneys, spleen, lungs, however, for the large majority of pharmaceutical drug metabolism occurs in the liver.
Therefore, assessment of in vitro intrinsic clearance or identification of metabolites formed is crucial at an early stage of the drug discovery process.

In vitro models for drug metabolism can include hepatocytes or subcellular fractions of the liver such as microsomes, cytosol, or S9 fraction, depending on the goal of the study. Hepatocytes are isolated directly from tissue; however subcellular fractions are obtained from tissue homogenate via centrifugation process (Fig.1). The way of subcellular fraction preparation results that microsomes are the simplest in-vitro metabolism model and contain only Phase I enzymes. A more complex approach takes into account the S9 fraction, in which Phase I and II enzymes are present. Hepatocytes are most representative because these contain both cell membrane as well as all enzymes for Phase I and II.  

Figure 1. The way of hepatocytes and subcellular fraction preparation

Within the liver, drug metabolism is achieved via two major enzyme reactions:

• Phase I reactions
• Phase II reactions.

Phase I enzymes include the cytochrome P450 (CYP) family of enzymes which are located in the smooth endoplasmic reticulum. The basic processes in phase I reactions are oxidation, reduction and/or hydrolysis, many of which are catalyzed by the CYP system and require NADPH as a cofactor. A final effect of this phase can be drug inactivation, prodrug activation to obtain a therapeutically active derivative, metabolite formation which is pharmaceutically active or toxic metabolite formation.

Phase II enzymes are located in the cytoplasm and endoplasmic reticulum and are characterized by conjugation reactions including glucuronic acid, glutathione, sulfate, and glutamine conjugations. Phase II reactions generally inactivate the drug if it is not already therapeutically inactive following Phase I metabolism, and make the drug more water soluble to facilitate its elimination.

Some drugs are metabolized by Phase I or Phase II enzymes alone, whereas others are metabolized by both Phase I and Phase II enzymes, so the choice of approach highly depends on the goal of the drug discovery process.

Metabolic Stability Assay

Selvita typically executes metabolic stability assays in mouse, rat, human microsomes, S9 fraction and hepatocytes (other species are on request). Test compounds are incubated at 37°C with a matrix in the presence or absence of enzyme cofactors. During this incubation, liver enzymes catalyze drug transformation. After the incubation, the reaction is terminated by the addition of cold acetonitrile. The loss of compound is determined by LC-MS analysis.

Protocol:

Exemple of Results:

Figure 1. The test compound decline with time in the presence of liver microsomes

Compound Requirements

COMPOUND REQUIREMENTS
1 mg or 20 µl of 10 mM DMSO stock

• Authors:
  Agnieszka Przybyłowicz, ADME/Bioanalysis Laboratory Manager
  Paulina Chęsy-Roman, Senior Analytical Specialist