Simultaneous quantification of abemaciclib and its active metabolites in human and mouse plasma by UHPLC-MS/MS
Alejandra Martínez-Chávez1, Matthijs M Tibben2, Karen A M de Jong1
1Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands; Division of Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
Abstract:
Abemaciclib is the third cyclin-dependent kinase 4 and 6 inhibitor approved for the treatment of advanced or metastatic breast cancer. In humans, abemaciclib is extensively metabolized by CYP3A4 with the formation of three active metabolites: N-desethylabemaciclib (M2), hydroxyabemaciclib (M20) and hydroxy-N-desethylabemaciclib (M18). These metabolites showed similar potency compared to the parent drug and were significantly abundant in plasma circulation. Thus, M2, M20, and M18 may contribute to the clinical activity of abemaciclib. For this reason, an UHPLC-MS/MS method for the simultaneous quantification of abemaciclib and its active metabolites in human and mouse plasma was developed and validated to support further clinical or preclinical investigations on this drug. Samples were processed by protein precipitation with acetonitrile, followed by supernatant dilution and filtration. Chromatographic separation was performed on a Kinetex C18 column (150 × 2.1 mm ID, 2.6 μm) using gradient elution with 10 mM ammonium bicarbonate in water (eluent A) and in methanol-water (9:1, v/v, eluent B). This method was selective, linear, accurate and precise within the range of 1-600 ng/mL for abemaciclib, 0.5-300 ng/mL for M2 and M20, and 0.2-120 ng/mL for M18. Furthermore, stability of the analytes in human and mouse plasma samples in several conditions was demonstrated. Finally, this assay was successfully used in a preclinical pharmacokinetic study, where abemaciclib and its active metabolites were identified and quantified. Inter-species differences between human and mouse samples were encountered, especially in the formation of M20, where isomers of this compound were detected in mouse plasma, but not in human plasma. This was confirmed by high resolution-mass spectrometry (HR-MS) measurements.
Insights
A new UHPLC-MS/MS method quantifies abemaciclib and its active metabolites in plasma. This assay supports preclinical studies, revealing inter-species differences in metabolite formation, particularly for M20.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Biochemistry
Background:
- Abemaciclib is a cyclin-dependent kinase 4 and 6 inhibitor for advanced breast cancer.
- Abemaciclib is metabolized into three active metabolites: N-desethylabemaciclib (M2), hydroxyabemaciclib (M20), and hydroxy-N-desethylabemaciclib (M18).
- These metabolites possess similar potency to abemaciclib and are abundant in plasma, potentially contributing to its clinical activity.
Purpose of the Study:
- To develop and validate a UHPLC-MS/MS method for simultaneous quantification of abemaciclib and its active metabolites (M2, M20, M18) in human and mouse plasma.
- To support further clinical or preclinical investigations of abemaciclib.
- To investigate inter-species differences in abemaciclib metabolism.
Main Methods:
- Sample preparation involved protein precipitation with acetonitrile, followed by supernatant dilution and filtration.
- Chromatographic separation was achieved using a Kinetex C18 column with gradient elution.
- Quantification was performed using UHPLC-MS/MS, with validated ranges for abemaciclib, M2, M20, and M18.
Main Results:
- The UHPLC-MS/MS method demonstrated selectivity, linearity, accuracy, and precision for abemaciclib and its metabolites across specified concentration ranges.
- Analyte stability was confirmed in human and mouse plasma under various conditions.
- The assay successfully quantified abemaciclib and its metabolites in a preclinical pharmacokinetic study, identifying inter-species differences, notably in M20 formation (isomers detected in mouse plasma only).
Conclusions:
- A validated UHPLC-MS/MS assay enables simultaneous quantification of abemaciclib and its active metabolites in human and mouse plasma.
- The method is suitable for preclinical pharmacokinetic studies.
- Significant inter-species differences in abemaciclib metabolism, particularly M20 formation, were observed between humans and mice.
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