A novel method was developed and validated for the quantification of the three approved CDK4/6 inhibitors (abemaciclib, palbociclib, and ribociclib) in both human and mouse plasma and mouse tissue homogenates (liver, kidney, spleen, brain, and small intestine) using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For all matrices, pretreatment was performed using 50 μL of sample by protein precipitation with acetonitrile, followed by dilution of the supernatant. Chromatographic separation of the analytes was done on a C18 column using gradient elution. A full validation was performed for human plasma, while a partial validation was executed for mouse plasma and mouse tissue homogenates. The method was linear in the calibration range from 2 to 200 ng/mL, with a correlation coefficient (
r
) ≥0.996 for each analyte. For both human and mouse plasma, the accuracy and precision were within ±15% and ≤15%, respectively, for all concentrations, except for the lower limit of quantification, where they were within ±20% and ≤20%, respectively. A fit-for-purpose strategy was followed for tissue homogenates, and the accuracy and precision were within ±20% and ≤20%, respectively, for all concentrations. Stability of all analytes in all matrices at different processing and storage conditions was tested; ribociclib and palbociclib were unstable in most tissue homogenates and conditions were modified to increase the stability. The method was successfully applied for the analysis of mouse samples from preclinical studies. A new ribociclib metabolite was detected in mouse plasma samples with the same
m/z
transition as the parent drug.
Electronic supplementary material
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Ribociclib
is a CDK4/6 inhibitor recently approved for the treatment
of some types of breast cancer in combination with an aromatase inhibitor.
It is currently investigated in the clinic to treat other malignancies,
including brain tumors. Using in vitro and genetically
modified mouse models, we investigated the effect of the multidrug
efflux transporters ABCB1 and ABCG2, and the drug-metabolizing CYP3A
enzymes on ribociclib pharmacokinetics and tissue distribution. In vitro, ribociclib was avidly transported by human ABCB1,
but not by human ABCG2 and only modestly by mouse Abcg2. Upon oral
administration at 20 mg/kg, the plasma AUC0–24h of
ribociclib was increased by 2.3-fold, and its terminal elimination
was delayed in Abcb1a/1b
–/–
;Abcg2
–/–
compared to wild-type mice. The brain-to-plasma ratios of
ribociclib were increased by at least 23-fold relative to wild-type
mice in Abcb1a/1b
–/–
;Abcg2
–/–
and Abc1a/1b
–/–
mice, but not noticeably in Abcg2
–/–
mice. Oral coadministration of elacridar,
an ABCB1 and ABCG2 inhibitor, increased the brain penetration of ribociclib
in wild-type mice to the same level as seen in Abcb1a/1b
–/–
;Abcg2
–/–
mice. Plasma exposure
of ribociclib further decreased by 3.8-fold when transgenic human
CYP3A4 was overexpressed in Cyp3a-deficient mice.
Ribociclib penetration into the brain is thus drastically limited
by ABCB1 in the blood–brain barrier, but coadministration of
elacridar can fully reverse this process. Moreover, human CYP3A4 can
extensively metabolize ribociclib and strongly restrict its oral bioavailability.
The insights obtained from this study may be useful to further optimize
the clinical application of ribociclib, especially for the treatment
of (metastatic) brain tumors.
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