Metabolic Stability

Metabolism

After a drug entering the body for a certain time, it will be eliminated either by excretion or degradation to one or more metabolites. The metabolic pathways of a drug can significantly affect its safety and efficacy, requiring dosing adjustments to achieve the safe and effective use of the drug.

Metabolism usually occurs in the liver and in vitro drug metabolism is measured by the activity of liver enzymes.

The aim of metabolic stability study is to measure the disappearance rate of a chemical compound. This study represents some of the earliest in vitro studies used in the pharmaceutical industry in an effort to predict in vivo pharmacokinetics. The concept of in vitro metabolic stability derives its importance from being related to metabolic clearance in vivo. This metabolic clearance in vivo has a relationship with drug bioavailability and half-life and hence gives information on how much and how frequently a drug should be administered.

Also, if a drug is slowly metabolized, the dose need to be adjusted and more preclinical toxicity tests should be conducted as long-time exposure may cause toxic build-up.

AMSbiopharma offers different biological systems to determine in vitro metabolic stability including primary hepatocytes and tissue fractions (e.g. microsomes and S9 fractions), using HPLC-MS/MS.

Hepatocyte Metabolic stability

Hepatocytes represent an independent in vitro cellular test system, containing both Phase I (modifications to the molecular structure itself) and Phase II (conjugation reactions) enzymes, cofactors and drug transporters, used to assess the metabolic stability of compounds and for early identification of species-specific differences.

AMSbiopharma hepatocyte stability assay monitors the disappearance of substrates in the presence of hepatocytes. In this assay, mixtures of test compound and pre-incubated hepatocyte suspension are incubated and removed at different time points and analyzed by HPLC-MS/MS to measure the test compound concentration.

Microsomal Metabolic stability

In early drug discovery, microsomes are typically the preferred system for measuring liver metabolism due to their relatively low cost, robustness and ease of use for high-throughput applications, low lot-to-lot variability due to pooling multiple donors, and wide availability from commercially reputable suppliers. Microsomes are prepared by homogenizing fresh or frozen livers with subsequent isolation of the endoplasmic reticulum subcellular fraction via centrifugation. A number of drug-metabolizing enzymes are located in the endoplasmic reticulum membrane, especially cytochrome P450 (CYP) and UDP-glucuronosyltransferases (UGT). 

In the AMSbiopharma standard microsomal metabolic assay mixtures of test compound and liver microsomes are incubated in the presence of NADH or VDFGA as cofactors. Aliquots are removed at different time points, stopped by quenching solution to precipitate the microsomal incubations. After centrifugation, a supernatant aliquot is analyzed by HPLC-MS/MS.

S9 Metabolic stability

S9 metabolic stability assay is very convenient for obtaining early knowledge about the metabolism of a new chemical entity. S9 represents the supernatant obtained by differential centrifugation at 9,000 g of liver homogenate and is rich in both microsomal and cytosolic fractions.

Liver S9 fractions include Phase I and II metabolic enzymes, are relatively inexpensive, easy to use, and amenable to automation, making them a more appropriate screening system. 

AMSbiopharma offers customized S9 stability assays to assess the metabolic stability of your candidate drugs.

 

Metabolic stability