In Vivo Impact Of MPEG_2k-PCL_x Polymeric Micelles On The Activity Of Hepatic CYP450 Enzymes In Rats

Purpose:In this study,mPEG_2k-PCL_x polymeric micelles with different molecular weights of hydrophobic segment(PCL_2k,PCL_3.5k,PCL_5k,PCL_7.5k,PCL_10k)were used to investigate their effects on activity of seven major CYP450 enzymes（CYP1A1/B2,CYP1A2,CYP2B1,CYP2C6,CYP2C11,CYP2D2,CYP3A1/2）,pharmacokinetics of CYP450 probe substrates and corresponding molecular biological mechanism after intravenous administration of these polymeric micelles in rats in order to elucidate the interaction between polymeric micelle-based nano-drug delivery system and comedication and provide the basis for reasonable design and application of micellar carriers.Methods:Firstly,a series of blank mPEG_2k-PCL_x micelles and DiR/coumarin6-loaded micelles were prepared by modified thin-film hydration method or nanoprecipitation method,followed by in vitro characterization with ZetaPlus Zeta Potential Analyzer,stability,and hemolysis assay.The uptake of micelles in isolated primary rat hepatocytes and biodistribution in rats were also investigated.Secondly,after intravenous administration at two dose levels of mPEG_2k-PCL_x micelles（5mg/kg,75 mg/kg）for 7 d and 14 d,respectively,the activity of CYP450 enzymes were exhibited by incubation with specific probe substrate and isolated rat liver microsomes（RLMs）followed by LC-MS/MS analysis,and the possible mechanism was further explored using real-time polymerase chain reaction（RT-PCR）,Western blotting analysis,and immunohistochemistry detection.Finally,mPEG_2k-PCL_3.5k.5k micelles with more obvious effects were chosen to evaluate the impacts on pharmacokinetic profiles of CYP probe substrate after intravenous administration with a single dose and multiple doses.Results:The results of characterization in vitro showed that the mean particle sizes of five mPEG_2k-PCL_x micelles varied from 20 to 100 nm with narrow size distributions（PDI<0.3）and the micelles were nearly neutral;micelles retained their particle sizes in the presence of FBS except for mPEG_2k-PCL_2k micelles,whose particle size increased to nearly two-fold after 1 h;the hemolysis percentage in all treated groups was less than 5%,indicating that mPEG_2k-PCL_x micelles showed non-hemolytic activities after intravenous injection.Five mPEG_2k-PCL_x micelles were able to enter into rat primary hepatocytes and mainly deposited in the liver with high retention at 48 h after intravenous administration in rats.The hepatic toxicity results showed that mPEG_2k-PCL_x micelles exhibited no significant effects on histopathology and the levels of ALT,AST,GSH,and ROS,indicating that the micelles had no obvious hepatotoxicity.With respect to the enzyme activity,CYP1A1/B2 was more susceptible to mPEG_2k-PCL_x micelles in comparison to other CYP isoforms,which was significantly induced or inhibited in most micelle-treated groups;mPEG_2k-PCL_3.5k micelles at 5 mg/kg increased the activities and mRNA levels of most CYP450 enzymes with enhanced inductive effects after administration for 14 d.However,polymeric micelles exerted less effect on protein expression except for CYP1A1 and CYP3A.The results of pharmacokinetic profiles of CYP probe substrates showed that treatment with a single dose of mPEG_2k-PCL_3.5k.5k micelles exerted obvious inhibitory effects or trends on the metabolism of most substrates,among which much stronger inhibitory effects on CYP1A2,CYP2C11 and CYP3A1/2 substrates were observed in high-dose group;consecutive administration with micelles at low dose for 7 d markedly induced the metabolism of substrates for CYP1A1/B2 and CYP2B1;after extending the duration of treatment to 14 d,mPEG_2k-PCL_3.5k.5k micelles showed enhanced inductive effects on CYP450 enzymes,among which the metabolism of phenacetin（CYP1A2）and omeprazole（CYP2C11）was more susceptible to be induced.Conclusion:mPEG_2k-PCL_x micelles were able to accumulate in the rat liver and enter into the hepatocytes to interact with CYP450 enzymes after intravenous administration,and the effects on metabolic function were highly relevant to the CYP450 isoforms,micelle types,administration duration and dose level.These results unveiled that nanocarriers showed potential inhibitory or inductive effects on rat hepatic CYP450 enzymes.Attention should be paid to the carrier-drug interaction when co-administration of these nanocarriers with other drugs metabolized by CYP450 enzymes.Moreover,appropriate carriers should be selected to evade the DDI-induced side effects.