| Polyethylene glycol?PEG?is a synthetic polymer comprised of repeating ethylene oxide subunits with molecular weight?MW?of 44 Da.PEG is widely used as excipients or form conjugates with drug molecules?PEGylation?and to prepare PEGylated drug delivery vehicles.When PEGs are used as excipients,or when PEGylated therapeutics release PEG in vivo through dissociation,leakage or breakdown,recipients of PEGs and PEGylated therapeutics are inevitably exposed to free PEG.Being neutral,non-toxic and of limited bioavailability due to their high hydrophilicity,PEGs have been approved for oral,intravenous and skin administration for over thirty years.There are numerous examples of the deleterious effects caused by PEG which include:accumulate in tissues,act as a P-gp inhibitor and produce an acid metabolite implicated in causing acidosis and hypercalcemia.Therefore,it is necessary to recognize and precision analysis of the mechanism of interaction between PEG and P-gp,polydispersity PEG in cells and the metabolism and dynamic process of the system level,clarify its toxic material base,reveal strong interaction mechanism between accessories-drugs,new drug delivery system to lay the foundation for scientific and reasonable design.Therefore,in this paper,we will study and discuss the following aspects.1.Mass spectrum analysis of PEGAn LC method was established for the separation of PEG homologues in the mass range of 400-2500 Da.Based on this method,triple triple quadrupole time-of-flight mass spectrometry?Triple-Q-TOF?was applied to investigate the charged and fragmented properties of PEG homologues.The ions with the highest response generated by PEG homologues are[M+nNH4]n+.The n depends on the MW of PEG homologues:MW<648.3843 Da,n=1;For 648.3843 Da?MW<1176.6902 Da,n?2;When Da?MW<1660.9672 Da,n?3;1660.9672 Da?MW<2145.2418 Da,n?4.When Da?MW<2500 Da,n?5,it can be concluded that PEG homologues with larger MW tend to carry more charges.As DP increases,PEG tends to carry less charge.When DP increased from 50 V to 100 V,the charge carried by the ions hardly changed.When DP increased from 100V to 150 V,the ions carrying 4 charges disappeared.When DP increased to 200 V,the ions carrying the 3 charges disappeared.The ions with 2 charges are most stable and does not change under different DP.Under the same CE condition,PEG with more charges are more likely to be fragmented,while the parent ion with 2 charges is not easy to break.The completion of this part of the study is conducive to the establishment of quantitative and qualitative methods in the next experiment,which is the methodological basis of the later experiments.The above methods were further developed to establish a method for the determination of low MW impurities?PEG750,2000 and 5000?in high MW PEG?20,000 Da?.2.The inhibition of PEG on P-gp in vitro and in vivo.PEG inhibited efflux of P-gp substrates such as rhodamine 123?Rho 123?,paclitaxel?PAC?and colchicine?COL?at low concentrations in the overexpressed P-gp Madin-Darby Canine Kidney-human multidrug resistance?MDCK-hMDR1?cells.The results showed that PEG inhibited P-gp.The effect of PEG on the pharmacokinetic behavior of PAC in rats indicated that PEG also inhibited P-gp in vivo.The interaction between PEG and P-gp is likely to lead to the excipient-drug interaction based on P-gp.Therefore,in the application of PEGylated drugs,it is necessary to pay attention to whether there is a P-gp substrate co-administered.3.The mechanism of PEG inhibit P-gp.It was found that the addition of two P-gp inhibitors,verapamil?VER?and cyclosporin A?CsA?,increased the accumulation of PEG with different MWs in the overexpressed P-gp Madin-Darby Canine Kidney-human multidrug resistance?MDCK-hMDR1?cells.It can be concluded that PEG of different MWs can be effluxed by P-gp.Once the drug is bound to the substrate binding site of P-gp,adenosine triphosphate?ATP?consumption and energy supply will inevitably result.Therefore,by detecting ATP consumption,it can be confirmed whether PEG is bound to the substrate site of P-gp.It was found that the addition of PEG with different MWs did lead to an increase in ATP consumption,which confirmed the conclusion that PEG binds to P-gp substrate binding sites.The above experiments have confirmed that PEG is the substrate of P-gp,so it can be concluded that the inhibition of PEG on P-gp is through competitive binding with the substrate binding site of P-gp.4.The mechanism of PEG entering cellsThe study found that the low MW PEG?PEG 750 and PEG 2000?entered the cell by passive diffusion,in which the lower MWs PEG homologues entered the cell more quickly,the uptake of the lower MW homologs is more rapid than uptake of the higher MW homologs.However,by 24 h,both profiles resemble those of the corresponding standard solutions.The high MW PEG?PEG 5000 and PEG 20,000?enters the cell enter cells by passive diffusion at low concentration and by a combination of passive diffusion and caveolae?mediated endocytosis at higher concentration.Moreover,with the increase of concentration,the proportion of endocytosis gradually increases.This is speculated to arise because PEGs 5000 and20,000 tend to aggregate at higher concentrations causing a higher proportion of their uptake to occur by endocytosis.These aggregated PEGs enter cells by endocytosis,others still enter cells by passive diffusion.Aggregation may also explain the weak stimulation of ATPase activity produced by PEG 20,000 at higher concentrations,aggregated PEGs could no longer interact with P-gp.It can be concluded that the low MW PEG enters the cell by passive diffusion,so it is easy to get out of cells by passive diffusion,which is why the low MW PEG is not easy to accumulate in the tissue.However,the high MW PEG may aggregate,which could not be expelled from the cells through passive diffusion after entering the cells,but could only be expelled from the cells through some energy dependence processes,so there is a risk of accumulation in the tissues.5.The polydispersity pharmacokinetic study of PEG in ratsThe results showed that only PEG 750 could be orally absorbed into animals,and the lower MW PEG homologues in PEG 750 had a higher oral absorption ratio.Through the study of the metabolism of PEG in vivo,it was found that PEG 750metabolized into hydroxyl acid metabolites in vivo,while PEG 2000 metabolized into PEG 750,PEG 5000 into PEG 500 and PEG 1000,and PEG 20,000 into PEG 500 and PEG 2000.Implementation of the proposed study is expected to provide pharmacokinetic data support for investigating the pharmacological and toxicological mechanism of PEGylated drugs,promote the approval of PEGylated drugs and provide evaluation basis for theirscientific design and safety and effectiveness study... |
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