Remdesivir, formerly GS-5734, has recently become the first antiviral drug approved by the U.S. Food and Drug Administration (FDA) to treat COVID-19, the disease caused by SARS-CoV-2. Therapeutic dosing and pharmacokinetic studies require a simple, sensitive, and selective validated assay to quantify drug concentrations in clinical samples. Therefore, we developed a rapid and sensitive LC-MS/MS assay for the quantification of remdesivir in human plasma with its deuterium-labeled analog, remdesivir-
2
H5, as the internal standard. Chromatographic separation was achieved on a Phenomenex® Synergi
TM
HPLC Fusion-RP (100 x 2 mm, 4 μm) column by gradient elution. Excellent accuracy and precision (<5.2% within-run variations and
<9.8% between-run variations) were obtained over the range of 0.5-5000 ng/mL. The assay met the FDA Bioanalytical Guidelines for selectivity and specificity, and low inter-matrix lot variability (<2.7%) was observed for extraction efficiency (77%) and matrix effect (123%) studies. Further, stability tests showed that the analyte does not degrade under working conditions, nor during freezing and thawing processes.
Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF–MEK–ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF–MEK–ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.
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