Preclinical Pharmacokinetic Study Of Lipid Nanoparticle Material JK-0315

Curretntly,the epidemic of COVID-19 has greatly changed people’s lifestyle,and even threatened people’s life and property safety,becoming a public health security event that has attracted worldwide attention.Based on the advantages of short development cycle and fast momentum of production capacity,the rapidly emerging m RNA vaccine has undoubtedly become the dawn of public health interventions when new infectious viruses are prevalent.However,the in vivo delivery of nucleic acids faces many limitations and challenges,such as enzyme degradation,cellular trafficking,and endosomal escape.Therefore,several strategies have been gradually developed to realize the safe and effective delivery of nucleic acid drugs.Among them,cationic lipid-based nanocarriers are the most efficient and least cytotoxic of non-viral nucleic acid delivery vehicles.In response to the urgency of severe situation of the COVID-19 epidemic,the LNP-based m RNA vaccine was approved for emergency use authorization,with large-scale vaccination carried out.It is worth noting that adverse reactions such as allergy and myocarditis have been gradually reported.As the core component of lipid nanoparticle,ionizable lipid usually accounts for more than 50%of the total lipid content.Relied on electrostatic interaction to load the nucleic acid,the in vivo distribution,degradation rate and metabolism of ionizable lipid will significantly affect the efficacy of vaccines,bringing potential risks of adverse reactions.Preclinical pharmacokinetic monitoring and characterization of novel ionizable lipids is of great significance,and will facilitate the design of safer and more effective m RNA-LNP vaccines in the future.In order to break through the technical monopoly and industry barriers of foreign LNP delivery of nucleic acid,domestic companies independently synthesized ionizable lipid JK-0315,which is an analog of Pfizer’s m RNA vaccine ionizable lipid,ALC-0315.According to the structural characteristics of ionizable lipids JK-0315,the biological sample pretreatment process of JK-0315 is constructed.Agilent ZORBAX300SB-C8 column with large diameter and short column length was selected to enable the elution of cationic lipids on the reversed-phase column.Subsequently,with the help of liquid chromatography-tandem mass spectrometry（LC-MS/MS）,a new method for accurate in vivo quantitative analysis of JK-0315 was developed.Methodological validation was performed as required by the guidelines.Ultimately,using this established method,the pharmacokinetic study of JK-0315 in rats has been carried out,revealing preclinical pharmacokinetic behavior of JK-0315 and providing data support for the design of safe and effective LNP-based nucleic acid drugs.（1）Pharmacokinetic study of JK-0315 in ratsThe LC-MS/MS quantitative analysis method of JK-0315 in rat plasma was established,and the method was confirmed according to relevant regulations.The results show that the method established in this chapter has good accuracy and precision in the range of 0.05-5 ng/m L,and can be used for the study of JK-0315plasma pharmacokinetics.The results of plasma pharmacokinetics showed that after intramuscular injection of JK-0315 in rats,T_1/2,AUC_0-t,Vd,CL,MRT_0-tand MRT_0-∞were 59.4±25.9 h,52.2±14.8 ng/L?h,125±60.3 L/kg,1.48±0.37 L/h/kg,51.0±4.13 h,94.9±26.0 h.The exposure of JK-0315 in the blood circulation system is small,and it is slowly eliminated from the body and continues to exist stably in the blood.After 5 days,the concentration of JK-0315 in plasma only drops to about a quarter of C_max.Compared with ALC-0315 whose half-life is 139 h,JK-0315 is relatively easier to clear in rats.The apparent volume of distribution（Vd）is much larger than the total volume of rat body fluids,indicating that JK-0315 has a low blood concentration and a large amount of it is distributed in tissues.Comparing the drug-time curves between male and female rats,it was found that the absorption and elimination of JK-0315 in males was faster,whose plasma concentration had a downward trend,but there was no statistical difference in the main pharmacokinetic parameters between female and male rats.（2）Biodistribution study of JK-0315 in ratsAn LC-MS/MS quantitative analysis method for JK-0315 in rat tissues was established,and the method was partially validated.Using the established method,the biodistribution of JK-0315 in the liver,spleen and muscle at the site of administration of rats on day 6 after intramuscular injection of 200μg/kg was investigated.JK-0315could be detected in the three tissues after 6 days of administration,and the average concentration of JK-0315 in the muscle of administration site was 113.5±175 ng/g.It can be seen that a large amount of JK-0315 residing in the administration muscle,only a slight amount enters the blood and distributes to other tissues.At the same time,a small amount of JK-0315 was exposed to rat liver.（3）Excretion study of JK-0315 in ratsThe LC-MS/MS quantitative analysis method of prototype JK-0315 in rat urine and feces samples was established,and the excretion study of JK-0315 in rats was carried out.The results showed that after intramuscular injection of 200μg/kg of JK-0315,no prototype JK-0315 was detected in rat urine,but only a small amount of prototype JK-0315 was detected in feces,and the cumulative excretion rate was less than one thousandth.Only a very small part of the prototype JK-0315 was excreted through the feces,which is speculated that most of the JK-0315 still resides in the muscle where it was administered.Moreover,the prototype JK-0315 may be metabolized and excreted in the form of metabolites.