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In vitro and in vivo metabolic profiling

Aim:

To obtain information on the number and type (phase I and phase II) of metabolites, the in vivo elimination routes and the abundance of metabolites produced in vitro and/or in vivo. Several species can be investigated by both approaches.

The extent of metabolic profiling studies can be highly variable and depends on the requirements of the Sponsor.

Test system/Species:

The in vitro metabolic profiling is generally performed in a system containing liver microsomes, specific systems or hepatocytes. Tests applying special systems, e.g. supersomes or specific isoenzymes are also possible. Only phase I metabolites will be detected in the samples of a microsomal model, while both phase I and phase II metabolites can be studied in samples originating from a system using hepatocytes.

For the in vivo testing generally rat as rodent and dog as non-rodent species are used.

Procedure:

In vitro metabolic profiling:

Both radio-labeled and non-labeled material can be applied for in vitro metabolic profiling tests. Metabolites of samples of non-labeled test item are analyzed by LC/MS/MS while those produced from radio-labeled material by HPLC with on-line radioactivity detection (HPLC/RAD). As for the quantitative relations (metabolite mass balance: abundance of the different metabolites) LC/MS/MS provides less accurate, while HPLC/RAD more accurate data (on molar basis). However, LC/MS/MS serves additional information concerning the molecular weight and structure of the metabolites produced.

In vitro tests are most suitable for the comparison of the species since microsomal preparations and hepatocytes of many species are available. Eventual species specific metabolites can also be detected.

In vivo metabolic profiling:

Radio-labeled material is administered to the test animals. The main excreta, i.e. urine, bile and feces and blood are collected and analyzed for total radioactivity. Representative samples are extracted and studied for the metabolites’ profile qualitatively and quantitatively.

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