Pharmacokinetic Study Of Polymer Pharmaceutical Excipient TPGS

The excipients contained in traditional preparations do not have significant pharmacological or toxicological effects in human,because they do not have biological activity and functions.However,nanocarrier drug delivery system（NDDS）rely on their polymer pharmaceutical excipients to maintain the structural integrity and thereby achieve the purpose of drug delivery and controlled release.Unlike excipients which are quickly cleared from the body,the dissolution（erosion）or degradation of polymer pharmaceutical excipients is a slow process.Therefore,polymer pharmaceutical excipients and their degradation products will inevitably have potential toxic effects in the interaction with the body.Interpreting the fate（pharmacokinetics,biodistribution,metabolism and excretion）of polymer pharmaceutical excipients and their degradation products in vivo is of great toxicological significance.However,due to the polydispersity of the molecular weight of polymer and the complexity of the biological samples,accurate quantitative analysis of polymer pharmaceutical excipients and their degradation products in vivo is very difficult.D-α-tocopheryl polyethylene glycol 1000 succinate（TPGS）is a widely used pharmaceutical excipient approved by the FDA for intravenous,oral and skin administration.As the emulsifier,solubilizer,P-glycoprotein and PEGylation modifier,TPGS is widely used in the development of nanocarriers.However,no pharmacokinetic studies of TPGS have be reported so far.Therefore,to explore the substance basis of potential toxicity and biosafety of TPGS in vivo,TPGS was used as the research object in this study.Based on the analysis strategy of in-source collision-induced dissociation（CID）and multiple reaction monitoring（MRM）,a bioassay for simultaneous quantitation of TPGS and its degradation product PEG1000in biological matrices was established by liquid chromatography tandem mass spectrometry（LC-MS/MS）and applied to pharmacokinetic study of TPGS in rats.The successful implementation of this project will enrich and develop the basic methods and theories of pharmacokinetics of polymers.It will also provide powerful technical support in pharmacokinetics for the development of TPGS-based NDDS with better biosafety.（1）Characterization of molecular weight and fragmentation behavior of TPGS in mass spectrometryIn this study,the molecular weight distribution of TPGS was characterized by Time-of-flight mass spectrometry（TOF MS）.By adjusting the declustering potential（DP）and collision energy（CE）,TPGS was prevented from being fragmented in the ion source and quadrupole collision chamber,which ensure that all different molecular weight components of TPGS could be detected with intact precursor ion and molecular weight distribution of TPGS was obtained.Moreover,we also analyze the charged state and adduct combinations of TPGS in the electrospray ion source.The results show that the molecular weight of TPGS used in this study showed a normal distribution with the mean molecular weight of about 1500 Da,which was basically consistent with the nominal value.In the electrospray ion source,TPGS mainly exists in the form of TPGS²⁺,and it is mainly added with 2NH₄.Then,the fragmentation behavior of TPGS in quadrupole collision chamber was analyzed by adjusting different CE.It was found that under the action of CE,TPGS could produce specific fragment ions m/z 513.3924（structure of vitamin E succinate）,m/z 557.4185（structure of vitamin E succinate connected to a PEG unit）and a series of fragment ions of low-polymerization PEG.Then,the fragmentation behavior of TPGS in ion source was investigated by triple quadrupole mass spectrometry.It was found that under the action of DP,TPGS could show the same fragmentation behaviors as in quadrupole collision chamber.This provided theoretical basis to analysis strategy of in-source CID and MRM for quantitation of TPGS.（2）Pharmacokinetic study of TPGS and its degradation product PEG1000 in ratsBased on in-source CID-MRM,a bioassay for simultaneous quantitation of TPGS and its degradation product PEG1000 in rat plasma was established by LC-MS/MS.We also performed method validation and applied this bioassay to pharmacokinetic study of TPGS administered by intravenous injection and oral administration to rats.After oral administration of TPGS to rats,TPGS and its degradation product PEG1000 were not detected in plasma.Free TPGS could not absorbed into systemic circulation or the bioavailability of TPGS was very low.It indicated that TPGS had a lower risk of toxicity in vivo when used as excipient for oral preparations.After intravenous injection of TPGS to rats,the apparent volume of distribution（Vd）of TPGS in rats was 3.1±1.17 L/kg,which was much larger than the total volume of rat’s entire body fluid of 0.667 L/kg.It was suggested that TPGS performed a wide distribution in rat and was likely to have specific binding or accumulation in some tissues and organs.The elimination half-life(T_1/2)and clearance（CL）of TPGS were 11.9±1.39 h and 0.178±0.047 L/h/kg respectively,indicating that TPGS was cleared slowly in rats.The main pharmacokinetic parameters of TPGS in female and male rats were not statistically different（P>0.05）.The peak concentration(C_max)of degradation product PEG1000 was 4.56±0.845μg/mL at approximately 10 min,and the system exposure(AUC_0-t)was 5.70±1.82μg/mL*h within 8 h.Compared with TPGS,its degradation product PEG1000had a shorter T_1/2（1.77±0.529 h）and was cleared faster in rats.（3）Biodistribution study of TPGS and its degradation product PEG1000 in ratsAn LC-MS/MS method based in-source CID-MRM for quantitation of TPGS and its degradation product PEG1000 in various tissues of rats was established.We also evaluated accuracy and precision,and applied this method to biodistribution study of TPGS and its degradation product PEG1000 in rats.The results showed that after intravenous injection of TPGS to rats,TPGS performed high concentrations in spleen,liver and lungs,and low concentrations in heart and kidney.The concentrations of TPGS in tissue with low blood flow perfusion rate was significantly lower than these in tissues with high blood flow perfusion rate.Higher concentration of TPGS was found in fat than muscle,which may be caused by the lipophilicity of vitamin E.Due to blood-brain barrier,TPGS was rarely distributed in brain.The biodistribution behavior of degradation product PEG1000 was basically the same as that of TPGS,except that the concentration of PEG1000 in kidney was significantly higher than that of TPGS.10 h after administration,the levels of TPGS and PEG1000were still high in spleen and were eliminated slowly in large intestine and small intestine,which suggested possible accumulation of TPGS and degradation product PEG1000 and potential organs toxicity after long-term administration.（4）Metabolism and excretion study of TPGS in ratsAn LC-MS/MS method for identification of TPGS and its degradation product PEG1000 in urine and feces of rats was established.Moreover,we also characterized molecular weight distribution of TPGS and its degradation product PEG1000 in urine and feces by comparison with reference substances.The results showed that after intravenous injection of TPGS to rats,TPGS was hydrolyzed by esterase in vivo to produce degradation product PEG1000.The degradation product PEG1000 would be degraded at a slow rate to produce PEGs with the lower degree of polymerization.The degradation product PEG1000 could be excreted in urine and feces,while TPGS could only be excreted in feces and not in urine.The study of molecular weight distribution found that the molecular weight distribution of TPGS and degradation product PEG1000 in excreta was basically the same as that of reference substances.It showed that the molecular weight distribution of TPGS and degradation product PEG1000 did not changed significantly in rats,and the all components with different molecular weight could be excreted in urine or feces.After 120 h of intravenous administration of TPGS,the total cumulative excretion of TPGS and its degradation product PEG1000 in urine and feces accounted for 73.7±17.3%of the TPGS dose.（5）Inhibitory effects of TPGS on human cytochrome P450 enzymesIn this section,the inhibitory effects of TPGS on human cytochrome P450enzymes was studied by hybrid probe substrates method.It was found that TPGS did not significantly inhibit CYP1A2 and CYP2B6 in the concentration range of0.222-444μM.According to the IC50 of TPGS,TPGS had very weak inhibitory effects on CYP2C8,CYP2C9,CYP2C19 and CYP2D6,and weak inhibitory effects on CYP3A4.However,as the pharmaceutical excipient,TPGS has a higher content in nanocarriers.Therefore,when the clinical dose of the loaded drug is administered,the concentration of TPGS in vivo may reach an effective inhibitory concentration on CYP450 enzymes.Therefore,when the TPGS-based NDDS are used in clinic,the plasma concentrations of TPGS and effective drugs should be monitored simultaneously to avoid excessive concentration of TPGS affecting activity of CYP450 enzymes,thereby changing efficacy and toxicity of drugs.In summary,in view of the gaps in the fate of polymer pharmaceutical excipient TPGS in vivo,this project established an LC-MS/MS method for the simultaneous quantitation of TPGS and its degradation product PEG1000 in biological samples,clarified the pharmacokinetics of TPGS and its degradation product PEG1000 and effects of TPGS on CYP450 enzymes,and developed the new idea and method for qualitative and quantitative analysis of polymer pharmaceutical excipients.These will provide theoretical basis for the development of TPGS-based NDDS,guide the scientific design of NDDS and improve its success rate of clinical translation.