In this study, we have constructed a highly specific, selective and sensitive LC-MS/MS method for the simultaneous quantification of neratinib and IS (sorafenib) in rat plasma (Figs. 1 and 2, Table 1–3). This validated LC-MS/MS assay was successfully applied to the pharmacokinetic determination of neratinib in rats. We for the first time demonstrated that pre-treatment with verapamil (a known P-gp inhibitor) resulted in significant increase in system exposure of neratinib in rats, as the value of Cmax and AUC0 − t increased and CL/F of neratinib decreased (Fig. 3 and Table 4). Further, we confirmed that P-gp was involved in the transport of neratinib using MDCK II and MDCK-MDR1 cells model (Fig. 4 and Table 5).
Neratinib is currently approved for the treatment of patients with early-stage and metastatic HER2+ breast cancer[1]. However, it is estimated that about 70% of HER2+ breast cancer patients are either innately resistant or acquire resistance to HER2-targeted drugs[15,16]. Abbas et al have identified neratinib as a sensitive substrate for CYP3A4 enzyme, and co-administration with CYP3A4 inhibitor caused significant increased system exposure of neratinib [15,17,18]. Therefore, increased CYP3A4 activity has been considered to be an important mechanism of neratinib resistance. In addition to the action of metabolic enzymes, drug excretion mediated by efflux transporters, overexpressed in tumor cells, also confers to the commonly known phenomenon of multidrug resistance against certain antineoplastic agents[19,20]. P-gp has been known to transport a wide range of drugs and xenobiotics from intra- to extracellular at many biological interfaces such as the intestine, liver, blood-brain barrier (BBB), and kidney. Hence, activity inhibition of P-gp may raise blood concentrations of its substrates. In fact, we have evidenced that pre-treatment with verapamil (an inhibitor of P-gp) significantly increased neratinib Cmax compared with administration of neratinib alone (Table 4), resulting in higher plasma exposure level of neratinib (Fig. 3).
To investigate the underlying mechanism of the effect of verapamil on neratinib disposition in vivo, MDCK cells transfected with the human MDR1 gene (MDCK-MDR1) cells were used to examine the influence of P-gp on the active transport of neratinib. In the intracellular accumulation assay, verapamil successfully inhibited the efflux of neratinib in MDCK-MDR1 cells (Fig. 4D). In transcellular bidirectional transport experiments, neratinib has been identified as a P-gp substrate according to the net flux ratio (> 2) (Fig. 4E and Table 5). Hence, induction or inhibition of P-glycoprotein can lead to drug–drug interactions of neratinib. Further, due to the overexpression of P-gp in tumor cells, the multidrug resistance (MDR) function of P-gp would affect the absorption and clinical therapeutic effect of neratinib, which may be a crucial mechanism for neratinib resistance.
Additionally, breakthroughs in cancer research have greatly improved the survival of patients with advanced breast cancer, however, the incidence of brain metastases has also increased accordingly [12,21]. Significantly, the HER2+ subtype harbored higher risk to metastasize to the brain than other breast cancer subtypes [21]. Neratinib, to a degree, has shown intracranial efficacy in treatment for HER2+ breast cancer patients with brain metastases. But many challenges remain as the barrier properties of BBB, resulting in the sub-therapeutic concentrations of chemotherapeutic drugs within the brain. P-gp is expressed at high levels in the capillary endothelial cells of the BBB, and activity inhibition of P-gp can result in increased concentration of substrate drugs [22–24]. Taken together, the therapeutic effect of neratinib in treating brain metastases may be improved when co-administrated with P-gp inhibitors.
Furthermore, the mechanism for neratinib efflux in BBB is not likely to involve P-glycoprotein, but rather may involve breast cancer resistance protein (BCRP), which is also located at the apical membranes of BBB[24,25]. These two transporters act cooperatively at the BBB, limiting the uptake of substrate drugs into the central nervous system (CNS) and affecting their pharmacokinetics, therapeutic efficacy, and safety[26,27]. In fact, we have investigated the role of BCRP on the transport of neratinib using MDCK II and stably transfected lines with human ABCG2 genes (MDCK-BCRP). However, the intracellular accumulation of neratinib in MDCK-BCRP cells showed no statistically significant difference with that in MDCK II cells. The addition of BCRP inhibitor (10 µM Ko143) did not alter the efflux of neratinib in MDCK-BCRP cells, suggesting that neratinib was probably not substrate for BCRP. Thus, the absorption of neratinib and its distribution in brain is considered mainly mediated by the activity or expression of P-gp protein.
MDCK cells belong to epithelial cell line of canine kidney origin, and are increasing used in evaluation of drug passive permeability and membrane transport studies, and easy to handle[28]. MDCK-MDR1 cells provides a suitable in vitro model for screening P-gp substrates, showing polarized overexpression of P-gp in the apical side[29,28]. The results obtained in MDCK cell well reflect the in vivo situation[26,29]. In addition, bidirectional transport assays with MDCK-MDR1 cells have been reported as a valuable in vitro assay to investigate if human P-gp prevents the test compound from crossing the BBB [30–32]. Therefore, the results obtained by MDCK II and MDCK-MDR1 cell lines can reveal the influence of P-gp on the access of neratinib to the CNS. However, the pharmacokinetics of neratinib in brain need to be further investigated in future studies.