A novel method could effectively identify serum metabolites of oligonucleotides (OGNs) used to treat patients with spinal muscular atrophy (SMA), according to a study.
The researchers set out to identify better methods for assessing serum samples of patients with SMA who were prescribed nusinersen (Spinraza®).
Using phenol/chloroform in a liquid-liquid extraction yielded suboptimal results, emphasizing that a purification phase with solid-phase extraction must also be part of the method, reported Sylwia Studzinska, of the Nicolaus Copernicus University in Torun, Poland, and colleagues.
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The findings were published online in the International Journal of Molecular Sciences.
Notably, the kind of amine in the stationary and mobile phases were the primary factors in the optimization of the procedure. Both impacted the accuracy of metabolite identification and separation selectivity, and the kind of amine also affected signal intensity, they found.
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These results show “how important separation is in the identification of OGN metabolites,” the researchers wrote.
“The careful development of sample preparation, OGN separation, and determination methods allowed for successful metabolite studies,” they added.
This experimental research analyzed the serum of patients previously diagnosed with SMA and treated with nusinersen, an oligonucleotide, to test various techniques for detecting the presence of this drug and its metabolites in peripheral blood.
The approach that showcased better detection capacity was microextraction by packed sorbents. Moreover, the kind of amine in the stationary and mobile phases highly influenced the results.
For example, the adequate identification of a metabolite relies on the selectivity of its separation, which is, in turn, determined by both of these parameters. The amine type further intervened with the signal’s intensity.
The study could then determine that both N,N-diisopropylthylamine and N,N-dimethylbutylamine reached the highest peak areas and resolution of separation with an octadecyl column with a terminal aryl group. This method allowed the researchers to identify more than 12 metabolites.
The enzymes responsible for most of the nusinersen metabolism were the 3’ and 5’ exonucleases. Unsurprisingly, evidencing 3’ and 5’ end shortmers easily occurred, along with other products of depurination and depyrimidination reactions.
Oligonucleotides, such as nusinersen, are a recent therapeutic alternative for treating SMA and other genetic diseases, and their use has been exponentially growing in the last few years. Although important advances continue to aid in understanding these drugs, many details regarding their behavior in the human body still need further research.
For example, “Differences in the levels and amounts of metabolites were observed between patient plasma, which may suggest differences in the metabolism of Spinraza depending on the child’s body,” the authors noted.
Reference
Studzińska S, Mazurkiewicz-Bełdzińska M, Buszewski B. Development of the method for nusinersen and its metabolites identification in the serum samples of children treated with Spinraza for spinal muscular atrophy. Int J Mol Sci. Published online September 05, 2022. doi:10.3390/ijms231710166
