Isolation, Characterisation, Simultaneous Quantification and In Vitro Cytotoxicity Assessment of Iguratimod and Its Degradation Products

ABSTRACT Iguratimod (IGU) is a novel cytokine inhibitor clinically approved for the treatment of rheumatoid arthritis. The structural features of the drug reveal several potential labile sites, including the sulphonamide group, formamide moiety and aryl–aryl ether linkage, all of which are susceptible to hydrolytic cleavage. The current study is undertaken with the objectives of developing a stability‐indicating high‐performance liquid chromatography (HPLC) assay for simultaneous quantification of IGU and its degradation products (DPs), characterisation and cytotoxicity assessment of the DPs. The present study investigates the forced degradation behaviour of IGU under various stress conditions. The DPs were separated and characterised by high‐resolution mass spectrometry and nuclear magnetic resonance spectroscopy. A possible degradation mechanism was proposed. A selective stability‐indicating HPLC assay was developed for the quantification of the drug and its DPs. In vitro cytotoxicity of the DP was assessed using the MTT cell viability and DNA ladder assays, whilst DNA damage markers were monitored by western blotting. The drug was susceptible to degradation under acidic and basic conditions, yielding two DPs. The acid DP exhibited a distinct capacity to compromise genomic stability. These results suggest that the conversion of IGU to its DP under acidic conditions confers significant DNA‐damaging properties. Given that the acid degradant is also a major metabolite of IGU, a thorough toxicological evaluation is warranted. Owing to the facile formation of the acid DP, the developed method can be applied to monitor IGU and its degradation during processing with acidic excipients and dissolution testing samples in an acidic medium. The method can also be modified to monitor IGU and acid degradants from plasma samples.