Reidentification Of Anti-NSCLC Activity Of Apatinib And Study Of Drug-drug Interaction Between Apatinib And Gypenosides

BackgroundLung cancer has ranked up as the most common malignant tumor worldwide due to the uprising incidence and high mortality,among which 80%-85% is defined as Non-small cell lung cancer(NSCLC),posing threat to human health in recent years.Considering that most patients are diagnosed in advanced stage of lung cancer,it is of great significance to develop individualized drug therapy for better prognosis.Apatinib is the first self-developed anti-tumor drugs of category 1.1 that has been on market since Dec.2014.The prototype of apatinib can effectively inhibit Vascular endothelial growth factor receptor-2(VEGFR2)to exert anti-cancer effects,and has been successfully applied in the treatment of gastric cancer with satisfactory safety and efficacy.In addition,the preclinical research and phase Ⅱ clinical trial showed remarkable anti-NSCLC activity,which could bring benefit to patients with high response rate and prolonged progression-free survival,indicating its prosperous clinic applications.However,problems still remained be solved: 1)The clinical efficacy of other VEGFR2 inhibitors in patients with lung cancer is significantly different from that of apatinib in clinical trials,suggesting that the anti-tumor mechanism of apatinib is not yet clear and needs further validation study;2)The high therapeutic cost lay above what patients can afford.Utilization of medical resources is relative low,and the clinical application is limited.Therefore,it is important to elucidate its mechanism of anti-tumor activity through target identification and to reduce the cost-effectiveness ratio,which is of great significance for promoting the individualized treatment of apatinib in clinic.High expression of protein kinase in tumor cells was reported to be in close relation with tumor invasion,migration and neovascularization,and therefore became the hotspot in anti-cancer research.Herein,apatinib was checked for potential influence on protein kinase activity,and v-Raf murine sarcoma viral oncogene homolog B1(BRAF)V600E mutein was designated as the potential target after a thorough consideration of the protein kinases in clinical practice.Furthermore,it was confirmed that drug exposure was closely related to drug effect,and explorations into the drug-drug interactions for pharmacokinetic alterations might act as an important approach to change drug exposure and improve effectiveness-cost ratio.In this study,we applied the BRAF HCC364 cell line with V600 E mutation in vitro and in vivo for target identification of apatinib.In the meanwhile,an animal model was established with gypenosides for potential influences on apatinib pharmacokinetic profiles to order to enhance apatinib system exposure as well as the effectiveness-cost ratio.Above all,this study aimed to promote individualized apatinib medication for rational clinical application in NSCLC patients.Methods and results1.Inhibitory effect of apatinib on proliferation of HCC364(BRAF V600 E mutation)and HCC827(BRAF wild)cell lines were assessed through Cell Counting Kit-8(CCK-8).HCC364 and HCC827 obtained with IC50 values at 84.1 nM and 1024.1 nM respectively,for which HCC827 exhibited a 12 times higher sensitivity than HCC364 after apatinib treatment,indicating that apatinib could better inhibit the proliferation of NSCLC cells harboring BRAF V600 E mutation.2.The effect of apatinib on apoptosis,cell cycle and migration activity of HCC364 cells was investigated,the cell was treated with co-incubated with apatinib(0,10,20 and 50 nM)for 48 h.It was confirmed that apatinib could block the HCC364 cell cycle in G1 phase,promote its apoptosis and inhibit the migration,and these effects were gradient dependent,suggesting its potential to prevent the BRAF V600 E mutated NSCLC from deterioration.3.Inhibition effect of apatinib on pathway associated with BRAF was assessed through Western Blotting.HCC364 cell was incubated with apatinib(50 nM)for 0,15,30,60 and 120 min,and Western Blotting analysis was applied to check the phosphorylation status of Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase(MEK)and Extracellular regulated protein kinases(ERK)located downstream of BRAF pathway.Results indicated a dramatic inhibitory effect of apatinib on the phosphorylation levels of MEK and ERK at incubation time for 30 min,confirming the inhibitory effect of apatinib on HCC364 cells through RAF/MEK/ERK pathways.4.Nude mice were inoculated subcutaneously with HCC364 cells to establish a tumor-bearing model in vivo.The inhibition effect of apatinib at different levels(32,65 and 100 mg/kg·d)and vemurafenib(250 mg/kg·d)on NSCLC was studied.It was observed in the results that both vemurafenib(250 mg/kg·d)and apatinib(32,65 and 100 mg/kg·d)could efficiently inhibit tumor growth with the inhibitory rate of 48.73%,51.58%,70.19% and 72.49% respectively.Apatinib also exhibit satisfactory inhibitory effect on NSCLC bearing BRAF V600 E mutation,which was gradient dependent,and even the effect of low dose of apatinib was superior to vemurafenib.In addition,vemurafenib and apatinib could effectively inhibit MEK and ERK phosphorylation through protein analysis in tumor cells;5.A HPLC-MS/MS method for determination of apatinib and its major metabolite(M1-1,M1-2,M1-6 and M9-2)in rat plasma was developed and fully validated in accordance with U.S.Food and Drug Administration(FDA)guidance(2013,Version 1)and Chinese Pharmacopoeia(2015 edition).The results of selectivity,linearity,accuracy,precision,dilution effect,extraction recovery and matrix effect met with the criteria of relevant regulations.Calibration was linear over the studied concentrations ranging from 1-2000 ng/m L.This method lay a solid foundation for pharmacokinetic study of apatinib;6.Influence of GP at different levels on the pharmacokinetic profiles of apatinib was explored.Results showed that co-administration of high dose of GP(400 mg/kg)with apatinib could significantly enhance the system exposure of apatinib as well as its metabolites,with AUC0-24 h of M0,M1-1,M1-2 and M1-6 increased by 3.53-,2.69-,2.53-and 1.90-fold,respectively.Additionally,high dose GP administration could reduce the conversion rate of apatinib.The above findings confirmed the synergistic effects of GP on apatnib metabolism and enhancement of overall system exposure.Conclusions1.The study had the first time discovered the selective inhibitory effect on NSCLC bearing BRAF V600 E,and elucidated the mechanism of anti-V600 EBRAF NSCLC activity of apatinib was through the inhibition of BRAF/MEK/ERK pathway,and was confirmed through in vivo experiment.In addition,apatinib showed superiority of anti-tumor activity over vemurafenib,a prevalent target medication.2.Gypenosides was confirmed of synergic effects with apatinib in a rodent model through drug-drug interaction study with improved drug exposure of apatinib as well as its major metabolites.The co-administration therapy could potentially reduce the cost of apatinib regim,providing a scientific basis for the combination therapy of apatinib in the future.