| At the end of 2019,the 2019 coronavirus disease（COVID-19）broke out in the world,which had a huge impact on the world’s medical care and economy.Comirnaty?is developed by Bio NTech-Pfizer,the first mRNA vaccine against COVID-19 approved by the U.S.Food and Drug Administration.The active ingredient mRNA is encapsu-lated in lipid nanoparticles（LNP）and delivered to target cells.LNPs are the current state-of-the-art mRNA delivery systems and typically consist of varying ratios of cho-lesterol,helper lipid,ionizable lipid and polyethylene glycol（PEG）-lipid.The excipient,ALC-0159,is a PEG-lipid in Comirnaty?vaccines,comprising a PEG moiety and a lipid moiety.Different from common inert excipients,PEG-lipid plays an important role in LNPs and affects various properties of LNP particles,including LNP particle size and transfection efficiency,etc.However,due to the unprecedented speed of the COVID-19 outbreak,a safe and efficient vaccine is urgently needed to curb the spread of the infectious disease.Therefore,the development time of the Comirnaty?vaccine is much shorter than that of traditional vaccines,and many studies on toxic and side effects have not yet been carried out,and the possible adverse reactions caused by the carrier materials need to be further explored.The excipient ALC-0159 was used for the first time in the Comirnaty?vaccine.Therefore,there is a lack of in-depth research on the material itself,and little is known about the excipient ALC-0159.So far,no related pharmacokinetic studies of ALC-0159 have been reported,its safety and in vivo fate are still unknown,and its metabolic behavior in vivo remains to be explored and ex-plained.Between December 2020 and August 2021,5805 cases of anaphylaxis and ana-phylactic shock were reported following approximately 560 million doses of Co-mirnaty?vaccine administered in the United States and Europe（approximately 10.44cases per million doses）.However,the cause of allergic reactions after Comirnaty?vaccination is still not definite.Given that allergic reactions caused by PEG have been reported in the literature,it is speculated that PEG-lipid ALC-0159 in Comirnaty?vac-cines is one of the potential allergic excipients.From this point of view,studying the behavior of ALC-0159 in vivo will provide certain theoretical support for analyzing the mechanism of vaccine adverse reactions,and at the same time provide a reference for the safety evaluation of the excipients themselves.In this experiment,PEG-lipid ALC-0159 was used as the research object,and a liquid chromatography-tandem mass spectrometry（LC-MS/MS）technique was used to develop an analytical method for the quantification of ALC-0159 in rat plasma,urine and feces by in-source collision-induced dissociation（CID）-Multiple reaction monitor-ing（MRM）was developed,elucidating the plasma pharmacokinetics and excretion be-havior of ALC-0159 in rats.（1）Analysis of mass spectrometry law of ALC-0159First,the molecular weight distribution and charged state of ALC-0159 were stud-ied by time-of-flight high-resolution mass spectrometry（TOF MS）.The method used is to set a lower declustering potential（DP）and collision energy（CE）to ensure that ALC-0159 is not broken in the ion source and quadrupole,and ALC-0159 was detected in its intact precursor ion state,so as to accurately characterize the mass distribution of ALC-0159.Results:The molecular weight of ALC-0159 was calculated from the mass-to-charge ratio and the number of charges carried.The molecular weight of ALC-0159was polydisperse,with a mean value of 2500 Da and a normal distribution,which was basically consistent with the theoretical molecular weight.In the electrospray ion source,most of the precursor ions of ALC-0159 carry 4 charges and 3 charges,and a few carry 2 charges.The adduct ions are[M+Na+2NH4+H]4+,[M+Na+NH4+2H]4+,[M+Na+NH4+H]3+and[M+Na+H]2+.Then,time-of-flight mass spectrometry was used to study the fragmentation be-havior of ALC-0159 in the Q2 collision cell,and a lower DP was set to ensure that ALC-0159 did not fragment in the ion source,and CE was set from small to large to explore the fragmentation of ALC-0159 behavior.The results showed that at CE 30 eV,the PEG part of ALC-0159 could generate fragment ions with m/z 89.0591,103.0753,133.0859,147.1015 and 177.1126,which provided a basis for the study of the in-source CID rule of ALC-0159,and laid a foundation for the selection of subsequent quantita-tive fragments.Next,the in-source CID rule of ALC-0159 in API 4000 triple quadrupole mass spectrometry was investigated.A larger DP was set,so that ALC-0159 was fragmented in the source,and suitable fragments were screened as parent ions to be fragmented again in Q2,and ALC-0159 had two collision-induced dissociation.The results show that ALC-0159 can produce the same fragmentation behavior as that in the quadrupole collision cell Q2 under the DP.This part of the study provides a prerequisite for the development of subsequent in-source CID-MRM quantitative methods.（2）Pharmacokinetic study of ALC-0159 in rat plasmaOn the basis of the previous stage,an LC-MS/MS method for the quantification of ALC-0159 in rat plasma by in-source CID-MRM was established,and the methodology was investigated.The results showed that the established method was reproducible and could be applied to the pharmacokinetic study of ALC-0159.Subsequently,the plasma pharmacokinetic behavior of ALC-0159 in rats was investigated.The results showed that,after intramuscular injection of 5 mg/kg ALC-0159:the peak concentration Cmaxwas 3.20±0.997μg/mL,the plasma drug concentration was still greater than one tenth of Cmax at 96 h after administration,the elimination half-life t1/2 was 130.902±60.671 h,the apparent volume of distribution Vd was 4.27±1.476 L/kg,the clearance rate was0.024±0.006 L/h/kg,the area under the concentration-time curve AUC（0-t）was181.061±36.533 mg/L·h,the AUC（0-）∞</sub>was 217.799±57.577 mg/L·h,the mean retention time MRT（0-t）was 67.279±5.663 h,and the MRT（0-）∞</sub>was 133.65±43.629 h,.After intramuscular injection of 10 mg/kg ALC-0159 in rats:the peak concentra-tion Cmax was 6.258±0.235μg/mL,the plasma drug concentration was still greater than one-tenth of Cmax at 96 h after administration,the elimination half-life t1/2 was103.7±29.2 h,the apparent volume of distribution Vd was 4.435±0.996 L/kg,the clear-ance rate was 0.03±0.003 L/h/kg,the area under the concentration-time curve AUC（0-t）was 287.849±36.533 mg/L·h,AUC（0-）∞</sub>was 334.008±27.131 mg/L·h,the mean reten-tion time MRT（0-t）was 71.108±1.586 h,and the MRT（0-）∞</sub>was 119.322±25.845 h.The results showed that the analyte ALC-0159 was eliminated very slowly in vivo,and there were individual differences among different rats.（3）Excretion study of ALC-0159An LC-MS/MS analytical method for the determination of ALC-0159 in rat feces and urine was established,and some methods were verified.The excretion of ALC-0159 in urine and feces was subsequently elucidated.The test results showed that the concentration of ALC-0159 in the urine sample was lower than the lower limit of quan-tification,ALC-0159 was excreted in the feces in its original form,and the cumulative excretion rate in the feces at 120 h was about 72.0%of the administered dose.In conclusion,high-resolution mass spectrometry TOF MS was used to qualita-tively analyze the analyte ALC-0159 in this experiment.Next,the in-source CID rule of ALC-0159 was analyzed in API 4000 unit mass-resolved mass spectrometry,and fragments with accurate molecular weights were found after assignment by high-reso-lution mass spectrometry,and the fragments with good stability and high response were screened for the establishment of LC-MS/MS analytical method.Finally,the plasma pharmacokinetics and excretion behavior of ALC-0159 in rats were investigated. |
PDF Full Text Request