Inhibitory Effects Of PEG With Different Degrees Of Polymerization On Human Liver Microsomal CYP Enzymes

Polyethylene glycol(PEG)is generally considered to be safe and nontoxic,and has unique physical and chemical properties as well as good biological compatibility.PEG with different degrees of polymerization had been approved by FDA.It was one of the very few synthetic polymers that can be injected in vivo,and widely used in the field of medicine.However,several studies showed that PEG was not an inert substance,and also could cause certain biological effects such as inhibition of CYP3A activity,induction of CYP3A expression and inhibiting the efflux pump P-glycoprotein activity etc.,which may influence the distribution and metabolism of drugs.PEG with different modified group or degrees of polymerization may have different properties,and the biological effects are also different.This study established human liver microsomal(HLMs)incubation system with cocktail probes based on the LC-MS/MS analysis method,and investigated the inhibitory effects of PEGs including PEG400,mPEG2K,mPEG5K,mPEG10K and mPEG20K on the eight primary CYP enzymes in HLMs.In this paper,LC-MS/MS analytical method was established for simultaneous determination of nine probes in HLMs incubation samples.The incubation samples were treated with chill methanol which contained melatonin as internal standard(I.S.)to precipitate proteins,and then the supernatant was diluted with methanol:water(1:1,v/v).The analytes were separated on a Agilent 300SB-C18 column using methanol-0.1%formic acid solution as mobile phase for gradient elution.Electrospray ionization(ESI)source was applied and multiple reaction monitoring(MRM)mode were used to quantify the analytes with positve and negative switching detection.All the compounds were analysed in a single run with 6 min.The validation test for three batches showed that the method was sensitive,accurate and reproducible,could be used for the quantitative determination of the metabolites in incubation samples.During this study,it was found that PEG can seriously affect the mass spectrometry signals of some analytes which were co-eluted.By monitoring the PEG ions m/z 133.1?89.1,chromatographic gradient was optimized.As a result,the four PEGs were separated from the metabolites,and only PEG400's retention time was close to part of the analytes.The impact of PEG400 on the determination of the metabolites was measured,and the datas were applied as references to correct the outcomes to avoid false positive or false negative results in incubation test.A cocktail probe incubation system was established to determine the eight kinds of CYP450 enzymes activity in HLMs and the probes were divided into two groups.Group ? contained PN 10 ?M,TS 10 ?M,TB 80?M,MPT 40 ?M,DMT 2.5 ?M and CZX 20?M to examine CYP1A2,CYP3A subfamily,CYP2C9,CYP2C19,CYP2D6 and CYP2E1 respectively;and group ? examined CYP3A subfamily,CYP2A6,CYP2B6,the coresponding probe substrates and their concentrations were respectively MDZ 2.5?M,CM 5 ?M and BPP 20 ?M.The concentration of HLMs protein was 0.2 mg/mL in the incubation system and the optimal concentration of NADPH was 5 mM and incubation time was 20 min for Group ? and 10 min for Group ? after optimization.The specificity of metabolic reactions in this incubation method was tested by comparing the CYPs' activities in cocktail substrates system angainst single substrate system,and results showed that the specificity of each probe metabolism in cocktail system was good,the interactions between the mixed probes could be ignored.The established cocktail probe incubation system was applied to investigate the Inhibitory effects on the 8 kinds of CYPs in HLMs by PEG400,mPEG2K,mPEG5K,mPEG10K,mPEG20K with the concentrations ranging from 0.1 ?M to 1 mM.When the PEGs' concentration reached 1 mM,all the CYPs' residual activities were greater than 80%when co-incubated with PEG400 or mPEG2K;and for mPEG5K the CYP enzymes' remain activites were greater than 90%except that the residual activity of CYP2B6 was 78.1%;mPEG10K and mPEG20K both inhibited more than 50%of the activity of CYP2B6,and greater than 45%of the activity of CYP 1A2 and CYP2C19 was inhibited by mPEG10K.The IC50 values of PEG with different degrees of polymerization on the CYP enzymes were really high relative to the inhibiiton end point refered in general rules,most of IC50 values were higher than 5 mM,but the IC50 was 1.26 mM for CYP1A2,1.18 mM for CYP2C19 and 1.06 mM of CYP2B6 which incubatied with mPEG10K,and the IC50 value of mPEG20K on CYP2B6 activity was 0.93 mM.The results showed that the inhibitory effect of PEG on the CYP enzymes was related to its degree of polymerization;compared to other CYP enzymes,the activity of CYP2B6 was more likely to be influenced by PEG.Overall,this study suggested PEG was a kind of material with negligible inhibition on CYP enzymes in HLMs,but as pharmaceutical excipient widely used,PEG could be administered with very high dose to reach the concentration that could inhibit CYPs' activity significantly which might result in drug interactions.