The influence of verapamil on the pharmacokinetics of the pan-HER tyrosine kinase inhibitor neratinib in rats: the role of P-glycoprotein-mediated efflux.

Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been approved for the treatment of HER2-positive (HER2+) early-stage and brain metastatic breast cancer. Thus far, the pharmacology effects and pharmacodynamics of neratinib have been well studied. However, the disposition of neratinib and its influencing factors in vivo remain unclear. P-glycoprotein (P-gp), one of the most extensively studied transporters, substantially restricts penetration of drugs into the body or deeper compartments (i.e., blood-brain barrier, BBB), regarding drug resistance and drug-drug interactions. Thereby, the aim of this study was to investigate the influence of verapamil (a P-gp inhibitor) on the pharmacokinetics of neratinib in rats. Here, we have established a high specific, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify plasma concentrations of neratinib in rats. Pharmacokinetic results showed that verapamil significantly increased the system exposure of neratinib, as Cmax increased by 2.09-fold and AUC0-t increased by 1.64-fold, respectively. Additionally, the in vitro transport of neratinib was evaluated using Madin-Darby canine kidney II (MDCK II) and human MDR1 gene overexpressed MDCK (MDCK-MDR1) cell line models. As a result, the net flux ratio was over than 2 and decreased over 50% by verapamil, suggesting that neratinib was a substrate of P-gp. Hence, our findings have highlighted the important role of P-gp in the system exposure of neratinib in vivo, and drug-drug interaction should be considered when coadministration of P-gp inhibitors with neratinib. These findings may support the further clinical development and application of neratinib.

O-Sulfation disposition of curcumin and quercetin in SULT1A3 overexpressing HEK293 cells: the role of arylsulfatase B in cellular O-sulfation regulated by transporters.

Extensive phase II metabolic reactions (i.e., glucuronidation and sulfation) have resulted in low bioavailability and decreased biological effects of curcumin and quercetin. Compared to glucuronidation, information on the sulfation disposition of curcumin and quercetin is limited. In this study, we identified that BCRP and MRP4 played a critical role in the cellular excretion of curcumin-O-sulfate (C-O-S) and quercetin-O-sulfate (Q-O-S) by integrating chemical inhibition with transporter knock-down experiments. Inhibited excretion of sulfate (C-O-S and Q-O-S) caused significant reductions in cellular O-sulfation of curcumin (a maximal 74.4% reduction) and quercetin (a maximal 76.9% reduction), revealing a strong interplay of sulfation with efflux transport. It was further identified that arylsulfatase B (ARSB) played a crucial role in the regulation of cellular O-sulfation by transporters. ARSB overexpression significantly enhanced the reduction effect of MK-571 on the cellular O-sulfation (fmet) of the model compound (38.8% reduction for curcumin and 44.2% reduction for quercetin). On the contrary, ARSB knockdown could reverse the effect of MK-571 on the O-sulfation disposition of the model compound (29.7% increase for curcumin and 47.3% increase for quercetin). Taken together, ARSB has been proven to be involved in cellular O-sulfation, accounting for transporter-dependent O-sulfation of curcumin and quercetin. A better understanding of the interplay beneath metabolism and transport will contribute to the exact prediction of in vivo drug disposition and drug-drug interactions.

Cinobufotalin inhibits the epithelial-mesenchymal transition of hepatocellular carcinoma cells through down-regulate ß-catenin in vitro and in vivo.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence and mortality. The prognosis of HCC is poor due to the high postoperative recurrence rate and metastasis rate. Epithelial-mesenchymal transition (EMT) plays a key role in the metastasis of HCC, which is closely related to the invasion, intrahepatic metastasis and low survival rate. Here we demonstrated that cinobufotalin can upregulate epithelial markers (E-cadherin) and downregulate mesenchymal markers (N-cadherin, snail, slug and ZEB1) in HepG2, SMMC-7721 and SNU-368 cells. We further found that the mRNA and protein expression of ß-catenin and its target genes (i.e. MMP7 and DKK1), which are related to tumor invasion and metastasis, were decreased after cinobufotalin treatment. Overexpression of ß-catenin promoted EMT of HepG2 and SMMC-7721 cells, and cinobufotalin could antagonize this induction. While Knockdown of ß-catenin could inhibit EMT and cinobufotalin enhanced this inhibition. In addition, cinobufotalin significantly suppressed the tumor EMT, as demonstrated by increased E-cadherin expression and decreased N-cadherin and vimentin expression, and inhibited formation and metastasis of lung metastases in vivo. In conclusion, our study has revealed a novel anticancer mechanism of cinobufotalin, which inhibits EMT progress by downregulating ß-catenin, and then prevents the migration and invasion of HCC. These results provide convincing evidence for the development of cinobufotalin as a potential HCC metastasis inhibitor.

HGF/c-MET pathway contributes to cisplatin-mediated PD-L1 expression in hepatocellular carcinoma.

Cisplatin has been reported to promote the expression of programmed cell death ligand-1 (PD-L1) in some cancer cells. However, the underlying mechanism through which PD-L1 is transcriptionally regulated by cisplatin in hepatocellular carcinoma (HCC) cells remains largely unknown. In the present study, we found that the expression of hepatocyte growth factor (HGF), p-Akt, p-ERK, and PD-L1 was increased in cisplatin-treated SNU-368 and SNU-739 cells. HGF stimulation also increased PD-L1 expression in these cells. Moreover, Inhibition of HGF/c-MET, PI3K/Akt, and MEK/ERK signaling pathways can dramatically block cisplatin or HGF-induced PD-L1 expression in SNU-368 and SNU-739 cells. In vivo, combination PHA665752 with cisplatin significantly reduced tumor weight with increased infiltration of CD8+ T cells in the tumor. Taken together, our study suggested that HGF/c-Met axis-induced the activation of PI3K/Akt and MEK/ERK pathways contributes to cisplatin-mediated PD-L1 expression. These findings may provide an alternative avenue for the treatment of HCC.