Determination of metarrestin (ML-246) in human plasma for a first-in-human clinical pharmacokinetic application by a simple and efficient uHPLC-MS/MS assay.

Metarrestin is a first-in-class small molecule inhibitor targeting the perinucleolar compartment, a subnuclear body associated with metastatic capacity. Promising preclinical results led to the clinical translation of the compound into a first-in-human phase I trial (NCT04222413). To characterize metarrestin's pharmacokinetic profile in humans, a uHPLC-MS/MS assay was developed and validated to determine the disposition of the drug in human plasma. Efficient sample preparation was accomplished through one-step protein precipitation paired with elution through a phospholipid filtration plate. Chromatographic separation was achieved with gradient elution through an Acuity UPLC® BEH C18 column (50 × 2.1 mm, 1.7 µm). Tandem mass spectrometry facilitated the detection of metarrestin and tolbutamide, the internal standard. The effective calibration range spanned 1-5000 ng/mL and was both accurate (range -5.9 % to 4.9 % deviation) and precise (≤9.0 %CV). Metarrestin proved stable (≤4.9 % degradation) under various assay-imposed conditions. Matrix effects, extraction efficiency, and process efficiency were assessed. Further, the assay was successfully able to determine the disposition of orally administered metarrestin in patients from the lowest dose cohort (1 mg) for 48 h post-administration. Thus, the validated analytical method detailed in this work is simple, sensitive, and clinically applicable.

Quantitation of the next-generation imipridone ONC206 in human plasma by a simple and sensitive UPLC-MS/MS assay for clinical pharmacokinetic application.

ONC206 is an imipridone derivative that is being developed clinically as a single agent given orally in a first-in-human trial (NCT04541082). This ongoing clinical trial requires pharmacokinetic analysis of ONC206 to fully characterize its pharmacologic profile. There is currently no published bioanalytical method for ONC206 quantitation. To understand the clinical pharmacokinetics of ONC206, a sensitive yet simple uHPLC-MS/MS method for quantitation of ONC206 in human plasma was developed. Protein-precipitation allowed rapid and sensitive bioanalytical measurement of ONC206 in human plasma. A Phenomenex Kinetex C18 (50 ×2.1 mm, 1.3 µm, 100 Å) analytical column achieved symmetrical and sharp chromatography peaks of ONC206 and the internal standard, [2H]7-ONC206, which were detected using multiple reaction monitoring. The assay calibration range was 1-500 ng/mL and was best fit by a linear regression model (r2 > 0.99732 ± 0.0010). The method proved accurate (< ± 9% deviation), precise (<11%CV), selective and specific with no interference and low inter-lot matrix variability. ONC206 demonstrated excellent short-term, long-term, and multiple freeze-thaw cycle stability in solution and human plasma. This fully validated method was used to quantitate ONC206 plasma concentrations from patients enrolled in the aforementioned clinical trial at the NCI to demonstrate its clinical applicability.

Vertical Inhibition of the RAF-MEK-ERK Cascade Induces Myogenic Differentiation, Apoptosis, and Tumor Regression in H/NRASQ61X Mutant Rhabdomyosarcoma.

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.

Ibrutinib's off-target mechanism: cause for dose optimization.

Ibrutinib (Imbruvica®, 2013) is a Bruton's tyrosine kinase (BTK) inhibitor approved for multiple B-cell malignancies and cGVHD. Its treatment is associated with increased risk of cardiac adverse events. Atrial fibrillation is a common cause of therapy discontinuation and interruptions, which have been correlated with shorter progression-free survival in chronic lymphocyte leukemia (CLL) patients. Recently, Xiao et al. identified that ibrutinib-mediated atrial fibrillation is likely due to off-target CSK inhibition. Given promising in vitro and in vivo evidence of maintained biological activity in CLL at lower-than-labeled ibrutinib doses, this elucidated mechanism substantiates the case to investigate alternative dosing schedules. The potential to minimize ibrutinib's off-target effects while conserving response warrants further discussion and investigation of optimal ibrutinib dosing.