Quantitation of navtemadlin in human plasma and brain tissue by liquid chromatography-tandem mass spectrometry.

Navtemadlin is an orally bioavailable small molecule that blocks the protein-protein interaction between murine double minute 2 protein (MDM2) and the tumor suppressor protein p53, leading to p53-mediated cell cycle arrest and apoptosis. It is being evaluated in clinical trials for a variety of malignancies, both as a single agent and in combination regimens. A sensitive, robust LC-tandem mass spectrometry (LC-MS/MS) method was developed to quantitate navtemadlin in plasma, and this method was also validated using brain tissue homogenate. Sample preparation involved protein precipitation of plasma or brain tissue homogenate using acetonitrile. Navtemadlin, navtemadlin glucuronide, and the internal standard, D6 -navtemadlin, were separated from microsomal incubation extracts using gradient elution and a ZORBAX XDB C18 column. Analytes were detected using a SCIEX 5500 triple quadrupole mass spectrometer in positive electrospray ionization mode. The assay range of 1-1000 ng/mL was shown to be accurate (96.1-102.0% and 95.7-104%) and precise (coefficient of variation ≤ 10.6% and ≤ 6.6%) in plasma and brain tissue homogenate, respectively. An 8000 ng/mL navtemadlin sample diluted 1:10 (v/v) with plasma was also accurately quantitated. Navtemadlin has been stable in frozen plasma at -70°C for at least 20 months. This validated LC-MS/MS method was applied to determine navtemadlin concentrations in plasma and brain tissue samples from two separate patients receiving 120 mg/day navtemadlin on protocol ABTC1604.

DNA methyltransferase inhibitor exposure-response: Challenges and opportunities.

Although DNA methyltransferase inhibitors (DNMTis), such as azacitidine and decitabine, are used extensively in the treatment of myelodysplastic syndromes and acute myeloid leukemia, there remain unanswered questions about DNMTi's mechanism of action and predictors of clinical response. Because patients often remain on single-agent DNMTis or DNMTi-containing regimens for several months before knowing whether clinical benefit can be achieved, the development and clinical validation of response-predictive biomarkers represents an important unmet need in oncology. In this review, we will summarize the clinical studies that led to the approval of azacitidine and decitabine, as well as the real-world experience with these drugs. We will then focus on biomarker development for DNMTis-specifically, efforts at determining exposure-response relationships and challenges that remain impacting the broader clinical translation of these methods. We will highlight recent progress in liquid-chromatography tandem mass spectrometry technology that has allowed for the simultaneous measurement of decitabine genomic incorporation and global DNA methylation, which has significant potential as a mechanism-of-action based biomarker in patients on DNMTis. Last, we will cover important research questions that need to be addressed in order to optimize this potential biomarker for clinical use.

Phase 1 study of belinostat and adavosertib in patients with relapsed or refractory myeloid malignancies.

PURPOSE: Belinostat is an intravenous histone deacetylase inhibitor with approval for T-cell lymphomas. Adavosertib is a first in class oral Wee1 inhibitor. Preclinical studies of the combination demonstrated synergy in various human acute myeloid leukemia (AML) lines as well as AML xenograft mouse models. EXPERIMENTAL DESIGN: This was a phase 1 dose-escalation study of belinostat and adavosertib in patients with relapsed/refractory AML and myelodysplastic syndrome (MDS). Patients received both drugs on days 1-5 and 8-12 of a 21-day cycle. Safety and toxicity were monitored throughout the study. Plasma levels of both drugs were measured for pharmacokinetic analysis. Response was determined by standard criteria including bone marrow biopsy. RESULTS: Twenty patients were enrolled and treated at 4 dose levels. A grade 4 cytokine release syndrome at dose level 4 (adavosertib 225 mg/day; belinostat 1000 mg/m2) qualified as a dose-limiting toxicity event. The most common non-hematologic treatment-related adverse events were nausea, vomiting, diarrhea, dysgeusia, and fatigue. No responses were seen. The study was terminated prior to maximum tolerated dose/recommended phase 2 dose determination. CONCLUSIONS: The combination of belinostat and adavosertib at the tested dose levels was feasible but without efficacy signals in the relapsed/refractory MDS/AML population.

ART714 is a best-in-class antileukemic 2-carbon-linked dimeric artemisinin derivative.

PURPOSE: It has become increasingly clear that new multiagent combination regimens are required to improve survival rates in acute myeloid leukemia (AML). We recently reported that ART631, a first-in-class 2-carbon-linked artemisinin-derived dimer (2C-ART), was not only efficacious as a component of a novel three-drug combination regimen to treat AML, but, like other synthetic artemisinin derivatives, demonstrated low clinical toxicity. However, we ultimately found ART631 to have suboptimal solubility and stability properties, thus limiting its potential for clinical development. METHODS: We assessed 22 additional 2C-ARTs with documented in vivo antimalarial activity for antileukemic efficacy and physicochemical properties. Our strategy involved culling out 2C-ARTs inferior to ART631 with respect to potency, stability, and solubility in vitro, and then validating in vivo pharmacokinetics, pharmacodynamics, and efficacy of one 2C-ART lead compound. RESULTS: Of the 22 2C-ARTs, ART714 was found to have the most optimal in vitro solubility, stability, and antileukemic efficacy, both alone and in combination with the BCL2 inhibitor venetoclax (VEN) and the kinase inhibitor sorafenib (SOR). ART714 was also highly effective in combination with VEN and the FMS-like tyrosine kinase 3 inhibitor gilteritinib (GILT) against MOLM14 AML xenografts. CONCLUSION: We identified ART714 as our best-in-class antileukemic 2C-ART, based on in vitro potency and pharmacologic properties. We established its in vivo pharmacokinetics and demonstrated its in vitro cooperativity with VEN and SOR and in vivo activities of combinations of ART714, VEN, and GILT. Additional research is indicated to define the optimal niche for the use of ART714 in treatment of AML.

A Novel 2-Carbon-Linked Dimeric Artemisinin With Potent Antileukemic Activity and Favorable Pharmacology.

Acute myeloid leukemia (AML) remains a devastating disease, with low cure rates despite intensive standard chemotherapy regimens. In the past decade, targeted antileukemic drugs have emerged from research efforts. Nevertheless, targeted therapies are often effective for only a subset of patients whose leukemias harbor a distinct mutational or gene expression profile and provide only transient antileukemic responses as monotherapies. We previously presented single agent and combination preclinical data for a novel 3-carbon-linked artemisinin-derived dimer (3C-ART), diphenylphosphate analog 838 (ART838), that indicates a promising approach to treat AML, given its demonstrated synergy with targeted antileukemic drugs and large therapeutic window. We now report new data from our initial evaluation of a structurally distinct class of 2-carbon-linked dimeric artemisinin-derived analogs (2C-ARTs) with prior documented in vivo antimalarial activity. These 2C-ARTs have antileukemic activity at low (nM) concentrations, have similar cooperativity with other antineoplastic drugs and comparable physicochemical properties to ART838, and provide a viable path to clinical development.