This technology includes a method of analyzing the potency of membrane transporter protein-based drugs acting on intracellular antioxidant and redox response pathways (and associated apoptosis pathways), wherein the drug delivery and activity is lipid associated. The present invention is a cell-based bioassay for measuring the bioactivity of drug substance and formulated drug product by determining the drug's dose-dependent inhibitory effects on 4 hydroxynonenal (4-HNE)-induced antioxidant response element (ARE) activity. The present invention covers use of HEK293 ARE-Luc Stable cell line as the detection system, high-throughput 96-well format, drug dilution approach, enclosure of negative controls with non-relevant heterologous proteins, use of the stimulator 4-HNE, and the optimal measurement time points with the ARE luciferase reporter gene. This is also an effective method to make liposome-drug substance mixtures for assembly of drug substance proteins into cellular membranes by using premade empty liposomes as a drug delivery system.
Applications include potency analysis of drug substance and drug product of membrane transporter protein-based drugs, such as RLIP76, acting on antioxidant and redox response pathways during the process, release, and stability monitoring.
This method standardizes the analysis of the potency of RLIP76-based drug substance and drug product in a dose-dependent manner during the manufacturing process, release, and stability monitoring of RLIP76 and similar molecules.