Study On The Pharmacokinetics Of Antitumor Compounds CYD1 And Piperine By LC-MS/MS

CYD1 is a dual HDAC and mTOR inhibitor,designed and synthesized independently by the research group,previous pharmacological studies confirmed its possession antitumor activity.Piperine is a pyrrolidine amide alkaloid obtained by extracting,separating and purifying the dried mature fruit of Piper nigrum L.In this study,both of CYD1 and the small monomeric compound Piperine were used as drug candidates for preclinical pharmacokinetic studies.The research includes three aspects:1.Study on In vitro metabolic stability and metabolic phenotype of CYD1 and Piperine.2.Study on the bioavailability of CYD1 and Piperine.3.Study on metabolites and metabolic pathways of CYD1in SD rats.The main contents are summarized as follows:1.1 Study on In vitro metabolic stability and metabolic phenotype of CYD1and PiperineUFLC-MS/MS technology was used to establish a method for simultaneous determination of CYD1 and Piperine in the in vitro liver microsome incubation system.The established method has good selectivity,high sensitivity and accuracy,good precision,no effect on residue,no matrix effect and good stability.The method conforms to the standard of biological sample analysis method and is suitable for simultaneous determination of CYD1 and Piperine in an in vitro liver microsome incubation system.1.1 In vitro metabolism of anti-tumor compound CYD1?1?The metabolic stability of CYD1 in different species of liver microsomes was studied.The results show that the in vitro metabolic half-lives of CYD1 in SD rat,Kunming mouse,human,rhesus and beagle liver microsomes are 110.00,33.32,52.50,83.49 and 77.87 min.It speculates that CYD1 is metabolized in human and Kunming mice,rhesus monkey,beagle dog liver microsomes,and metabolized slowly in SD rat,indicating that CYD1 is similar in human,SD rat,Kunming mice,There are species differences in the metabolism of rhesus monkey and beagle dog liver microsomes.In the subsequent selection of in vivo experimental animals,Kunming mice,rhesus monkey and beagle dog,which are closer to human metabolism,can be considered as a reference.?2?Using the selective chemical inhibitor method to study the phenotype of CYD1.The results show that CYD1 has no specific metabolic phenotype and is not susceptible to adverse effects due to specific metabolism.1.2 Study on In vitro metabolism of Piperine?1?The metabolic stability of Piperine in different species of liver microsomes was studied.The results show that in vitro metabolism half-life of Piperine in liver microsomes of SD rats,Kunming mice,human,rhesus monkey and beagle dog are 48.46,138.60,31.36,147.45 and 165.00 min respectively.It speculates that Piperine is metabolized similarly in human and SD rat liver microsomes,and metabolism is relatively fast.The metabolic rate is similar in the Kunming mice,rhesus monkey and beagle dog liver microsomes,and they are all slower.It indicates that there are significant species differences in the metabolic rate of Piperine in human,SD rat,Kunming mice,rhesus monkey and beagle dog liver microsomes.In the subsequent selection of in vivo experimental animals,SD rat that is more closely related to human metabolism can be considered as a reference.?2?Using the selective chemical inhibitor method to study the phenotype of Piperine metabolism,the results show that the enzymes involved in the metabolism of Piperine in human liver microsomes mainly include CYP3A4 and CYP2C9.Studies of metabolic phenotypes can predict their potential drug-drug interactions.It is suggested that the interaction between Piperine and other drugs should consider the interaction between drugs metabolized by CYP3A4 and CYP2C9.2.Study on the bioavailability of CYD1 and PiperineUFLC-MS/MS technology was used to establish a method for simultaneous determination of CYD1 and Piperine in body plasma samples.The method has good selectivity,high sensitivity and accuracy,good precision,no effect on residue,no matrix influence and good stability.The method conforms to the standard of biological sample analysis method and is suitable for simultaneous determination of CYD1 and Piperine in plasma.2.1 The bioavailability study of CYD1The results of bioavailability study on CYD1 show that the bioavailability of CYD1 is extremely low.The body half-life of CYD1 is very short after intravenous administration?T1/2=0.23h?,and the in vivo retention time(MRT_?0-t?=0.28h)is also short,indicating that CYD1 is rapidly eliminated in vivo.2.2 The bioavailability study of PiperineThe results of the bioavailability study on Piperine show that the bioavailability of Piperine is 59.95%,indicating that oral administration can be considered in later clinical studies.3.Study on metabolites of anti-tumor compound CYD1 in SD ratsA single dose of CYD1 was intravenously administered to each Sprague-Dawley?SD?rats?n=4?.The plasma,urine and feces samples were collected before and after administration,respectively.The all samples were subjected to ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry?UPLC-Q-TOF-MS/MS?analysis and for the identification of metabolites.A total of 13 metabolites except the prototype drug CYD1 were found,of which 3 I phase metabolites and 10 phase II metabolites.The results indicate that CYD1 is mainly eliminated in feces by phase II metabolism.Acetylation,methylation and redox are the main metabolic pathways.