Investigation on photo-isomeric impurity of Roxadustat: Structure conformation, physicochemical characterization, interconversion feasibility and in vitro toxicological evaluation

This research article describes the comprehensive characterization and toxicological activities of a photoisomeric impurity (PI) of an antianemic drug Roxadustat (ROX). The PI, which has the same molecular mass as that of the drug, possesses strikingly distinct structural features compared to the drug. A detailed investigation on structural as well as toxicological aspects of this impurity is warranted due to likelihood of formation of this impurity because of routine light exposure of the API, and due to alerts concerning to its carcinogenicity, mutagenicity, and teratogenicity based on in silico assessments reported in the literature. The molecular structure of the PI was confirmed by single crystal XRD providing the first evidence for the unambiguous structure determination. Complete physicochemical characterization of the impurity was carried out using powder X-ray diffraction, Fourier Transform Infrared Spectroscopy, Differential Scanning Calorimetry, and Thermogravimetric Analysis establishing phase identification and phase purity of the impurity. The feasibility of interconversion of API (ROX) to the impurity PI and vice versa was evaluated using the RP-HPLC and Triple Quadrupole LC-MS techniques for both the solution state as well as solid state. The in vitro toxicological activity of the PI was assessed through cytotoxicity assays, DNA fragmentation assays, morphological analysis, and COMET assay. These structural, synthetic and toxicological studies contribute to the overall understanding of drug stability, safety, and pharmaceutical formulation development of ROX.