Reactive Metabolite Analysis

BOC Sciences is engaged in a wide variety of metabolic research. Relying on a professional technical team, we provide reactive metabolite analysis services for evaluating and detecting reactive metabolites of drugs.

Reactive Metabolites

After most drugs enter the body, they are converted into inactive products through in vivo metabolism and excreted. However, some drugs are converted into active metabolites and reactive metabolites under the action of various metabolic enzymes after entering the body. Among them, reactive metabolites refer to the reactive intermediates formed after the drug is converted by metabolic enzymes. These metabolites are usually electrophilic and can bind to nucleophiles (eg, glutathione, cysteine) in the body, or covalently bind to biomacromolecules (eg, proteins, DNA) to produce toxic reactions. Studies have shown that reactive metabolites are involved in many idiopathic adverse drug reactions (IADRs). Therefore, in the early stage of drug screening and development, identifying the production of reactive metabolites is of great significance for the toxicity assessment of candidate drugs and timely adjustment of research strategies.

Our Service

As part of the overall evaluation of the absorption, distribution, metabolism and excretion (ADME) properties of candidate drugs, BOC Sciences uses a variety of in vivo and in vitro screening methods to analyze whether there are reactive metabolites, and identify the properties of reactive metabolites to avoid surprises Toxicity caused by specific drugs.

We add small molecule nucleophiles to capture reactive metabolites in vitro and generate stable metabolites. Suitable nucleophilic capture reagents include glutathione (GSH) and its derivatives, cyanide, semicarbazides, and the like. The structures of these conjugates were then detected and identified by high-resolution accurate mass spectrometry, ultimately enabling the screening and analysis of reactive metabolites.

Chemical Capture Reagents

• In vitro liver microsome incubation
• Forms stable adducts with many active substances
• GSH and its derivatives (such as reduced GSH, N-acetylcysteine (NAC), glutathione ethyl ester, etc.)
• Amines (e.g., semicarbazides, methylsemaline)
• Cyanides (such as potassium cyanide)
• Synthetic Peptide Capture Reagents

Mass Spectrometry Methods

• Neutral loss scanning
• Precursor ion scanning
• Multiple reaction monitoring (MRM)
• Mass defect filter (MDF)
• Background subtraction
• Liquid chromatography-tandem mass spectrometry (triple quadrupole, ion trap, quadrupole-linear ion trap, high-resolution mass spectrometer)
• Time-of-Flight Mass Spectrometry (TOFMS)

Time- and Cofactor-Dependent Inhibition of CYP450

In some cases, bioactivation of CYP-induced drugs leads to the formation of reactive metabolites. Reactive metabolites bind irreversibly to the active center of the enzyme, resulting in loss of enzyme activity. This mechanism-based inhibition requires the presence of NADPH co-actors and is a time-dependent process. Thus, in vitro time- and cofactor-dependent CYP inhibition responses allow detection of reactive metabolite production. We offer CYP inhibition assays to analyze reactive metabolites.

• Drug candidate co-incubated with liver microsomes
• Add CYP isozyme pattern substrate
• Quantitative detection by LC-MS/MS or fluorescence method

Toxicity test

BOC Sciences provides toxicity testing services to evaluate the toxicological potential of drug reactive metabolites, ultimately leading to early stage lead optimization of drugs.