The evaluation of inhibitive effectiveness of the tumour necrosis factor-α converting enzyme selective inhibitors by HPLC

Abstract

A novel high-performance liquid chromatography (HPLC) method based on the internal standard method was established for assaying the tumour necrosis factor-α converting enzyme (TACE) activity and matrix metalloprotease-9 (MMP-9) activity, and was used to evaluate the inhibitive effectiveness of inhibitors to TACE and MMP-9. In the assay method for TACE and MMP-9, peptides labelled with the ultraviolet group-Dpa were used as substrates. Alanine-Dpa was synthesised and was used as the internal standard for quantitative analysis. After the peptide substrates were hydrolysed by TACE (MMP-9) for 15min (25min) at 37°C, the amount of remaining substrates were determined by reversed-phased HPLC with UV detection at 353nm. The relative peak area of the substrate was linearly dependent on the substrate concentration. This method was then applied to determine the 50% inhibitory concentration (IC₅₀) of GM6001 and inhibitor A for both TACE and MMP-9.

Keywords::

• High-performance liquid chromatography
• internal standard method
• tumour necrosis factor-α converting enzyme
• inhibitive effectiveness