| Background and purposeDrug targeting therapy has come to be a hot research area in recent years,sorafenib,a multi-kinase inhibitor drug,has a dual function of inhibiting tumor cell proliferation and angiogenesis and is used as first line treatment for hepatocellular carcinoma and advanced renal cell carcinoma.But,there was a high incidence of adverse reactions and the treatment could not be sustained in clinical therapy.Sorafenib mainly undergoes CYP3A4-mediated oxidation and UGT1A9-mediated glucuronidation in liver,recent studys also showed no increase in sorafenib exposure following the concomitant administration of the highly potent CYP3A4 inhibitor ketoconazole,so,there should be other CYP isoforms or ways contribute to the SRF metabolism,and the biotransformation of sorafenib in kidney remains unclear.In this study,sorafenib metabolism and the expression of genes determined by RNA sequencing were evaluated in non-tumor kidney samples from Chinese patients.On the one hand,we can reveal sorafenib metabolism in kidney.On the other hand,we also need to find some important metabolic enzymes and transcription factors involved in the biotransformation of sorafenib.These studies can provide important evidence for clinical medication and metabolic mechanism of sorafenib.Methods and contents1.We collected human liver and kidney tissues from HCC and RCC patients who had undergone surgery resection.Kidney and liver microsomes were prepared by differential centrifugation,and we extracted total RNA by the Trizol method2.Kinetic parameters were determined in HKM and HLM firstly.Secondly,the phase I metabolism of sorafenib was evaluated in 36 individual non-tumor kidney samples.Several recombinant CYP isoforms were used to catalyze sorafenib for the final certification.3.The phase I metabolism of sorafenib and the expression of genes determined by RNA sequencing were evaluated in 36 individual non-tumor kidney samples.Real time PCR experiments were conducted to validate the RNA sequencing results.4.Kinetic Analysis and Statistical Analysis.We used the XL-Kinetics program to determine the best-fit model.Kinetic parameters were obtained according to the profile of Eadie-Hofstee plots.The comparison of metabolic rate involved in the thesis was using two independent sample T test for statistical analysis.Results1.Kinetics of sorafenib oxidation in HKM and HLMM2 and M3 were the principal metabolites in pooled human liver microsomes,whereas,M3 were the principal metabolites in SRF oxidation in pooled human kidney microsomes.The Km value of M3,M4 and M9 in pooled HLMs(85.286±16.667 ?M,71.374±25.355 ?M and 112.370±21.491 ?M)was 3-fold higher than that in pooled HKMs(27.014±12.847 ?M,16.439±15.764 ?M and 46.263±19.360 ?M).2.The correlation between the RNA sequencing and sorafenib metabolismA total of 19812 genes passed the QC(average of count? 5,FPKM>0 in each sample)filters and were included in the correlation analysis.The mRNA levels of CYP4F11(r=0.710,p=1.74×10-6),CYP4F2(r=0.620,p=7.24×10-5)and CYP4A11(r=0.459,p=0.006)were significantly correlated with the formation rate of M3 while CYP3A4(r=0.388,p=0.021)and HNF4A(r=0.419,P=0.012)showed moderate correlation.3.Recombinant human CYPs and Real time PCR experimentSorafenib was catalyzed by recombinant human CYP3A4,CYP4A11 and CYP4F2,two metabolites(M3 and M9)were observed in CYP4F2 and CYP4A11,The Km value of M3 were 5.423±5.455 ?M in CYP4F2 and 25.189±17.178 ?M in CYP4A11.PCR results showed that the RNA sequencing result is reliable(p<0.01).Conclusions1.The correlation analysis between RNA-Seq and metabolic activities can effectively identify the important enzymes as well as some important TFs involved in drug metabolism2.M3,M4 and M9 were detected in sorafenib oxidation in HKM and M3 was the principal metabolite.M9,a new sorafenib oxidized metabolite,remains to research in future.CYP4F2 and CYP4A11 contributed to the biotransformation of sorafenib in kidney.3.CYP4F11 and CYP4F2 Can be used as a predictor for sorafenib metabolic rate in the kidney and that can be used to guide individualized clinical medication... |
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