Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy

Abstract

The application of oligonucleotides as drugs for different genetic diseases is increasing rapidly. Since 2016 they are used during spinal muscular atrophy treatment with the use of nusinersen oligonucleotide. The purpose of this study was to improve methods for the analysis of serum samples of patients treated with nusinersen. The results showed that liquid-liquid extraction (with phenol/chloroform) is insufficient and an additional purification step using solid-phase extraction is necessary. The best results were obtained for microextraction by packed sorbents. Important parameters in the optimization of the method were mainly the type of amine in the mobile phase and the stationary phase. Both influenced the selectivity of metabolite separation and thus their correct identification; while amine type impacted also the intensity of signals. Finally, the highest resolution of separation and the highest peak areas were obtained for N,N-dimethylbutylamine or N,N-diisopropylthylamine with an octadecyl column with a terminal aryl group. Over a dozen of metabolites were successfully identified with the use of methods developed during the study. The 3′ exonucleases and 5′ exonucleases were mainly responsible for nusinersen metabolism, consequently, 3′end shortmers, and 5′end shortmers were observed, as well as metabolites with simultaneous loss of bases at both ends of the sequence. However, some depurination and depyrimidination products were also identified. To the best of our knowledge, this is the first report on nusinersen and its metabolite identification in serum samples by liquid chromatography and mass spectrometry.

Study of nusinersen metabolites in the cerebrospinal fluid of children with spinal muscular atrophy using ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry

“…Hybridization is, therefore, not an effective method for extracting nusinersen metabolites from blood plasma. 9,15…”

“…Ion-pair reversed-phase liquid chromatography (IP-RP-HPLC) is used most widely to separate metabolites of oligonucleotides, while MS allows their successful identification in the case of nusinersen. [9][10][11][12][13] Oligonucleotide analysis typically employs ion pair chromatography with alkylamines. The original ion-pairing reagent was triethylammonium acetate (TEAA), but nowadays, fluoroalcohols are used more commonly instead of acetic acid.…”

“…6,14 Nusinsersen metabolites in extracts from serum samples were analyzed by LC-MS with mobile phases consisting of mixtures of HFIP with N-dimethylbutylamine (DMBA), N,N-diisopropylethylamine or dipropylamine and methanol. Utilization of DMBA allowed partial separation of 15 metabolites within an 11-minute timeframe with the highest signal intensities (among all three amines) in MS. 9 To analyse nusinersen metabolites in serum samples, appropriate sample preparation is necessary, which, according to the reviewed publications, most often involves first liquid-liquid extraction (LLE) with a phenol/chloroform mixture followed by an additional purification step with one of the following techniques: solid phase extraction (SPE), hybridization, micro-extraction by packed sorbent (MEPS), and dispersive solid phase extraction. 9,15,16 Research of serum samples from children treated with nusinersen clearly indicates that using the SPE method resulted in the highest metabolite recoveries, demonstrating the effectiveness of this technique.…”

“…Hybridization is, therefore, not an effective method for extracting nusinersen metabolites from blood plasma. 9,15 Nusinersen undergoes metabolism through the activity of 3′ and 5′ endonucleases. Degradation of successive phosphodiester bonds between nucleotides occurs, and shorter fragments of nusinersen circulate in the bloodstream and are excreted in the urine.…”

Scientific Discoveries Supporting Theories in Science: From Thinking to Practice

“…The other two articles reported data on rare diseases. The first, from Studzi’nska, S et al [ 23 ], investigated the role of treatment with oligonucleotides in spinal muscular atrophy, a genetic disease. They used nusinersen oligonucleotide to detect both nusinersen and its metabolite in the serum of treated individuals.…”