Therapeutic oligonucleotide (nusinersen) metabolism in cerebrospinal fluid samples of patients with spinal muscular atrophy based on liquid chromatography coupled with mass spectrometry data

Abstract

Background: Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disorder caused by a deficiency of the survival motor neuron protein. The introduction of antisense oligonucleotide therapy has markedly improved prognosis, particularly following approval of Nusinersen (Spinraza), the first SMA drug. Although modified with 2′-O-methoxyethyl and phosphorothioate groups, nusinersen is metabolized. Comprehensive characterization of these metabolites in cerebrospinal fluid is limited due to analytical challenges associated with antisense oligonucleotides. This study aimed to develop a first liquid-liquid/solid-phase extraction procedure combined with ion-pair ultra-high-performance liquid chromatography coupled with mass spectrometry for the extraction, separation, and identification of nusinersen and its metabolites in cerebrospinal fluid samples from SMA patients treated with Spinraza. Results: A two-step sample preparation procedure provided high nusinersen recovery (89.2±1.8%), repeatability, eliminated matrix effects, and enabled 50-fold sample concentration. All of these proved essential for the detection and identification of low-abundance metabolites collected four months after dosing. Reliable metabolite identification requires sufficient chromatographic resolution, especially for metabolites differing by a single nucleotide with similar nominal m/z values. Consequently, careful ion-pair reagent selection and MS optimization are crucial for improving separation and sensitivity to detect low-abundance metabolites. Our results showed that propylamine and dimethylbutylamine may be used interchangeably, but the second one provides higher resolution. Accurate mass measurement and characteristic fragment ions derived from methylated nucleobases and phosphorothioate groups ensured reliable identification. Analysis of CSF samples from 17 pediatric SMA (at various dosing stages) patients revealed extensive in vivo metabolism, predominantly via 3′-exonucleolytic cleavage, yielding multiple N-shortmers. For the first time, interpatient variability was observed in nusinersen detectability and metabolite profiles of CSF samples. Significance: To our knowledge, this is one of the first comprehensive studies demonstrating the applicability of the developed procedure to observe differences in the metabolism of nusinersen and, in the future, linking them to therapeutic effects, therapeutic monitoring, or the patient’s condition. The methodology addresses key bioanalytical challenges (reproducibility, purification, concentration, separation) and enables metabolite profiling in a complex biological matrix. Moreover, it provides a foundation for future investigations linking metabolism, drug exposure, and clinical response in SMA therapy.