The LC-MS/MS assay was applied for DMB metabolic stability assessment with an RLM matrix, and the two parameters, (157.5 min) and (0.97 mL/min/kg), were calculated. linearity of the established LC-MS/MS assay ranged from 2 to 500 ng/mL with 0.9999. The limit of detection (t1/2 (0.97 mL/min/kg) and intrinsic clearance (157.5 min). Such values infer that DMB would be excreted very slowly from the human body, which might lead to possible bioaccumulation. To the best of our knowledge, this is the first method for DMB analysis in RLMs with metabolic stability estimation. Introduction Lung cancer is the leading cause of death among all cancer types, in particular, non-small cell lung cancer (NSCLC) is considered the most widespread [1C5], with an incidence of approximately 90%. The epidermal growth factor receptor (EGFR) signaling pathway has gained importance in the last few years as a therapeutic target for NSCLC [6]. Tyrosine kinase inhibitors (TKIs) that control EGFR are very efficient in the treatment of cancers possessing EGFR mutations, with a characteristic therapeutic window. First-line TKIs controlling EGFR (e.g., erlotinib and gefitinib) have good initial responses against these mutations [7, 8]. Unfortunately, acquired resistance in ~60% of patients and toxicities that occur during treatment [9, 10] decrease their therapeutic efficacies [11, 12]. This has led scientists to develop second-generation, irreversible EGFR TKIs (e.g., dacomitinib (DMB) L-cysteine and avitinib) [13, 14]. DMB (Fig 1) overcomes the acquired resistance observed with first-line EGFR TKIs [13C15]. It was shown to improve progression-free survival when compared with that of gefitinib in the treatment of NSCLC patients with positive EGFR mutations. This represents a new achievement for the treatment of these patients [16]. On September 27, 2018, the Food and Drug Administration (FDA) approved DMB in the form of VIZIMPRO tablets for the first-line treatment of patients with metastatic NSCLC harboring EGFR exon 19 deletions or exon 21 L858R substitution mutations [17]. In addition, a DMB marketing authorization application was accepted by the European Medicines Agency (EMA) for the same indication [18]. Open in a separate window Fig 1 Chemical structures of dacomitinib and Mouse monoclonal to AURKA lapatinib (IS). To the best of our knowledge, a single LC/MS-MS assay was lately published reporting the analysis of DMB in rat plasma [19]. The purpose of the present study was to establish a validated LC-MS/MS assay to quantify DMB in rat liver microsomes (RLMs) as a different biological matrix to the drug and to allow the application of this assay to investigate the DMB metabolic stability by calculating two important parameters (i.e., intrinsic clearance and half-life (t1/2)). These parameters could then be utilized for t1/2, hepatic clearance, and bioavailability calculations. Bioavailability is important because it provides information about the metabolism of the investigated compound; if the compound is rapidly metabolized, it will exhibit low bioavailability [20]. Experimental Reagents and chemicals All chemicals and solvents were of analytical grade. Dacomitinib (DMB) and lapatinib (internal standard; LTP; IS) were purchased from Med Chem Express (Princeton, NJ, USA). Rat liver microsomes (RLMs), Acetonitrile (ACN), ammonium formate (NH4COOH), and formic acid (HCOOH) were purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC-grade water (H2O) was obtained from the Milli-Q plus L-cysteine filtration system (Millipore, Billerica, MA, USA). LC-MS/MS methodology All LC-MS/MS parameters were optimized to achieve the best chromatographic resolution of DMB and IS with good separation. LTP was chosen as the IS in the DMB analysis because the same extraction procedure can be applied efficiently with a great success for both compounds L-cysteine (DMB and LTP recoveries were 97.913.74% and 97.2 1.3%, respectively in the RLM matrix) and the elution time of LTP is comparable to that of DMB. The proposed procedure is rapid with 4 min run time. Both LTP and DMB are TKIs and are not co-administered to patients, so this assay might be.
The LC-MS/MS assay was applied for DMB metabolic stability assessment with an RLM matrix, and the two parameters, (157
