Quantitation of Tuvusertib (M1774) in Human Plasma by LC-MS/MS

Abstract

Ataxia-telangiectasia and Rad3-related (ATR) protein kinase is an essential regulator of the DNA damage response (DDR) at stalled and collapsed replication forks. Tuvusertib (M1774) is a selective, orally available small molecule ATR inhibitor currently in preclinical and clinical development for cancer treatment. This study presents a robust and simple 5-min assay designed for the quantification of single agent tuvusertib in human plasma utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS). A 20 μL volume of plasma was subjected to protein precipitation, followed by chromatographic separation using a Phenomenex Synergi Polar-RP (4 μm, 2.1 × 50 mm) and a gradient mobile phase system consisting of 0.1% formic acid in both water and acetonitrile during a 4-min run time. Mass spectrometric detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, our assay met the criteria outlined by the Food and Drug Administration guidance for bioanalytical method validation, demonstrating robust performance within the range from 5 to 5000 ng/mL. This assay will support ongoing and future clinical studies by defining tuvusertib pharmacokinetics.

Keywords: LC‐MS/MS; M1774; human plasma; pharmacokinetics; tuvusertib; validation.

FIGURE 1 |

Representative chromatograms of (A) tuvusertib (m/z 371.2 > 179; 0.7 min) added to control human plasma at the LLOQ concentration of 5 ng/mL (top trace with an offset of 20 × 10³ counts) and control human plasma (bottom trace) and (B) tuvusertib-IS (m/z 375.2 > 183; 0.7 min) added to control human plasma at a concentration of 50 ng/mL (top trace with an offset of 40 × 10³ counts) and control human plasma.

FIGURE 2 |

Tuvusertib plasma concentrations in patients administered 90 mg of tuvusertib PO with (left panel) and without (right panel, with geometric mean, SD) accidental proton pump inhibitor (PPI) use.