Overcoming bioanalytical challenges during the development of risdiplam for the treatment of spinal muscular atrophy.

Aims: Risdiplam is a small molecule approved for the treatment of spinal muscular atrophy (SMA). The drug and its major metabolite had to be measured in plasma and tissue from several animal species and in human plasma and urine. Bioanalytical challenges including light sensitivity, instability, carryover, nonspecific binding, and complex tissue analysis, had to be overcome.

Materials & methods: Liquid chromatography tandem mass spectrometry with reversed-phase separation after protein precipitation/dilution was applied. Ascorbic acid was used as a stabilizer to mitigate degradation of the metabolite, and a surfactant additive prevented nonspecific binding in urine. Tissues were efficiently homogenized by bead beating and matrix-matched with plasma.

Results and conclusions: The above challenges were successfully addressed with bioanalytical methods tailored to study needs. Validations and regulatory analyses met requirements of current guidelines, including successful incurred sample reanalysis (ISR) in GLP and clinical studies. The 3R principles (Replacement, Reduction, Refinement) were applied in animal studies to minimize the use of real matrices. Pediatric studies were supported with rapid analysis and microsampling. Bioanalysis supported patient-centric approaches in dose finding and sampling and was key in answering important questions to enable risdiplam to the market.