Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

Abstract

Aim: Imetelstat, a 13-mer oligonucleotide with a lipid tail is being evaluated for treating hematologic myeloid malignancies. This report describes the development of extraction and quantification methods for imetelstat. Methodology & results: Imetelstat was extracted using SPE (rat plasma) or by hybridization using a biotinylated capture probe (human plasma) and was quantified by LC-MS/MS. Calibration curves were established (0.1-50 μg/ml). Stability of imetelstat in plasma was demonstrated. Concentrations of imetelstat extracted using either of the methods and quantified with LC-MS/MS were comparable with a validated ELISA. Conclusion: Two extraction methods (solid phase and hybridization) were developed for quantifying imetelstat in plasma using LC-MS/MS. The hybridization extraction in combination with LC-MS/MS is a novel extraction approach. In recent years, oligonucleotide drugs (ODNs) have gained popularity owing to their ability to target specific genes and provide specificity. ODNs comprise an oligonucleotide sequence, containing 10-50 nucleotides [1,2]. Numerous ODNs have been investigated so far [1][2][3][4]. But only six ODNs (fomivirsen, pegaptanib, mipomersen, eteplirsen, nusinersen and defibrotide sodium) have been approved by the US FDA since 1998 [5].Pharmaceutical development of ODNs has several technical challenges such as maintaining in vivo stability, ensuring delivery and bioavailability, and minimizing off-target effects. These challenges can be minimized by chemical modifications of ODNs to enhance their pharmacokinetic and pharmacodynamic properties [4,6]. These chemical modifications include: sulfurization of the phosphodiester bond to avert degradation by enlindogenous exonucleases, addition of methoxy or methoxyethyl groups to sugar moieties [7,8], locked and unlocked nucleic acids, and alterations in the internucleotide linkage (e.g., amide linkage) resulting in peptide nucleic acids [1]. Sulfurization results in phosphorothioate analogs that are more hydrophobic, exhibit more complex secondary structures with higher protein binding and greater accumulation in organs than phosphodiester analogs [9][10][11].Imetelstat is a novel, first-in-class telomerase inhibitor ODN [12], being investigated for the treatment of hematologic myeloid malignancies [13,14] and certain solid tumors [12,15,16]. Imetelstat is a 13-mer oligonucleotide N3 -P5 thio-phosphoramidate (NPS) with a covalently linked C16 (palmitoyl) lipid moiety at the 5 -end (Figure 1; Supplementary Table 1). Addition of a lipid chain and the modified oligonucleotide backbone enables imetelstat to penetrate cells and tissues, with high biodistribution into normal and malignant cells [12].Imetelstat differs from antisense oligonucleotides in its mechanism of action. Imetelstat is complementary to the template region of the RNA component of telomerase, to which it binds with high affinity and specificity, and directly competes with telomere binding, thereby inhibiting telomerase activity [17]. Thus, imetelstat acts as a classical ac...

Hybridization Liquid Chromatography–Tandem Mass Spectrometry: An Alternative Bioanalytical Method for Antisense Oligonucleotide Quantitation in Plasma and Tissue Samples

“…The optimized LC method achieved satisfactory separation of ASOs but suffered from a steady drop of sensitivity, which was significant in long batches and interday comparisons. This observation has been reported in other studies without any viable solutions, which has been one of the critical issues that limited the application of LC–MS/MS for ASO bioanalysis for many years . Though freshly replaced aqueous mobile phase reversed the signal intensity to the original level immediately, the sensitivity drop started soon after hours of aging, indicating potential stability or solubility issue with the aqueous mobile phase.…”

“…Chromatographic assays, typically liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), have been exploited as alternatives to LBA for ASO bioanalysis. − The workflow usually involves liquid–liquid extraction (LLE), solid-phase extraction (SPE), or both, followed by LC–MS/MS analysis. − With higher specificity as the major advantage, LC–MS/MS methods can distinguish and quantitate truncated metabolites in addition to the full-length ASOs. , However, low sensitivity, labor-intensive extractions, and lack of robustness prohibit LC–MS methods from being formally validated or widely used in drug development …”

“…To overcome the limitations of HELISA and LC–MS/MS, both techniques were combined into a hybridization LC–MS/MS method that extracted ASOs using hybridization and analyzed them using LC–MS/MS. This strategy was first reported by Dillen et al in 2017, when they established an initial method reaching 0.1 μg/mL sensitivity in plasma and raised questions for metabolite specificity . They also reported an intrarun decline in response when using typical ion-pairing (IP) LC–MS/MS conditions, which has been a common issue that essentially limits the application of LC–MS/MS for scaled-up ASO sample analysis in preclinical and clinical studies.…”

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

“…The peptide of ORN84 is not modified to convey stability against hydrolases. Therefore, we examined the stability of the SSOs in a plasma stability assay that mimics conditions in the bloodstream (48,49). We incubated the SSOs in mouse plasma and removed aliquots periodically for analysis by liquid-chromatography mass spectrometry (LC-MS).…”

Characterization of the In Vitro Inhibitory Potential of the Oligonucleotide Imetelstat on Human Cytochrome P450 Enzymes with Predictions of In Vivo Drug-Drug Interactions

“…Imetelstat (13-mer oligonucleotide N39-P59 thio-phosphoramidate with a covalently linked C16 [palmitoyl] lipid moiety at the 59 end) is in clinical development as a treatment option for hematologic myeloid malignancies (Röth et al, 2010;Baerlocher et al, 2015;Chiappori et al, 2015;Tefferi et al, 2015;Dillen et al, 2017). Imetelstat is a highaffinity, active-site telomerase inhibitor with specificity for the RNA template region of telomerase, directly competing with telomere binding.…”