Metabolic Stability

Purpose

The metabolic stability of a drug candidate is an important consideration in determining its potential for human use. The metabolism of the drug in the body, the half-life of the drug in the body, and whether it forms metabolites are important parameters to assess the bioavailability, toxicity, and dosing potential for drug-drug interaction of a compound.

Ricerca's metabolic stability assays are designed to quickly determine whether a compound is metabolized by hepatic drug metabolizing enzymes. Adding time points to the assay allows the determination of the metabolic rate, measured as the "intrinsic" clearance rate. Coupled with permeability data, these assays can weed out early those compounds with unacceptable pharmacokinetic properties. Same site in vivo pharmacokinetic studies, medicinal chemistry, radiolabel synthesis, LC-MS/MS, and metabolite ID expertise are available to confirm bioavailability and clearance predictions and identify metabolites.

Assay Protocol

Hepatocytes and microsomes from a variety of species are used to study the metabolic stability of the test article.

Hepatocytes

The test articles are incubated with previously cryopreserved hepatocytes, at 37°C in a 5% CO₂ atmosphere. Portions of the incubation medium are removed at various timepoints for up to 4 hours. In addition, appropriate positive and negative control incubations are performed.

The % turnover of the test article is determined utilizing LC-MS/MS.

Microsomes

The test articles are incubated with liver microsomes (typical protein concentration of 0.5 mg/mL) and 1 mM NADPH in phosphate buffer at 37°C for various timepoints up to 2 hours. The incubations are initiated by the addition of the microsomes and quenched by the addition of an equal volume of methanol. In addition, appropriate positive and negative control incubations are performed.

The % turnover of the test article is determined utilizing LC-MS/MS.

Confirmation Data

Data is typically generated using human and rat hepatocytes as well as human liver microsomes. As an example, data shown below is a metabolic stability experiment using testosterone as the test article.

Metabolism of Testosterone in Human Hepatocyte Cultures