Preliminary Study On The Stereoselectivity Of Praziquantel And Its Analogue P96 In Pharmacokinetics And Metabolic Mechanisms

Schistosomiasis is a serious hazard of zoonotic parasitic diseases of humans and animals. Schistosomiasis japonica is an epidemic disease in China. As the drug of first choice for the treatment of the disease, praziquantel (PZQ) has strong effect to kill adult schistosome, while poor effect to the larvae. At present, PZQ is the exclusive drug for the treatment of schistosomiasis japonica. The result is unimaginable if PZQ has drug resistance. Therefore, the research on new drug which could be used to treat schistosomiasis has important significance. In recent years, pharmacokinetic stereoselectivity of chiral compounds has become an important and indispensable topic of chiral drugs in drug development. Compared with racemic drug, enantiomer has many advantages:good therapeutic benefit, weak side effect and high safety. Therefore, the research of enantiomer drug is now one of the focus of the new drug innovation direction. World Health Organization (WHO) proposed a project to take place of rac-PZQ by R-PZQ for the treatment of schistosomiasis.P96 is a new compound with structure modification from PZQ. It has a pharmacodynamic activity in the treatment of schistosomiasis, not only good therapeutic effects on all kind of schistosomiasis, but also remarkable activity for schistosomiasis japonicum imagoes and larvae. Hence, P96 can be the research topic for the treatment of schistosomiasis. If candidate compounds P96 can be used as medicine, more choice can be provide for the treatment of schistosomiasis (especially schistosomiasis japonica). As a chiral compound, P96’s stereoselectivity is also worthy of attention.PZQ is metabolized by cytochrome P450 3A4 (CYP3A4). This enzyme plays a crucial role in the first-pass effect and bioavailability. Pregnane X receptor (PXR) is a main nuclear receptor mediating CYP3A4 induction. So, the research of PXR is very important for the interpretation of metabolic mechanism. Reporter gene assay is used to investigate the CYP3A4 induction by measuring the activated abilities of the compounds to PXR.In this paper, we carried out a primarily study on the pharmacokinetic stereoselectivity and metabolic mechanisms of PZQ and P96. The main research contents and results are as follows:1. Adopted a LC-MS/MS method to detect concentration in rat plasma of PZQ (R-PZQ, S-PZQ, rac-PZQ) or P96 (R-P96, S-P96, rac-P96) after oral administration. The differences between pharmacokinetic stereoselectivity could be found by comparing their pharmacokinetic parameters. The results showed that AUC, Vz, CLz, Cmax of R-PZQ and S-PZQ had significant differences (P<0.05). AUC、Vz、CLz、Cmax of R-PZQ and rac-PZQ had significant differences. But, for rac-PZQ and S-PZQ, the significant difference in Cmax was only found. AUC, MRT, t1/2, Tmax, Vz, Cmax of R-P96 and S-P96 had significant differences. AUC, MRT, t1/2, Vz, CLz, Cmax of R-P96 and rac-P96 had significant differences. Compared rac-P96 with S-P96, the significant differences in AUC, MRT, Tmax were found. The research results could provide the reference for substituting rac-PZQ by R-PZQ and the development of new compound P96.2. The Dual-luciferase reporter gene assay was used to investigate the stereoselectivity of PXR by PZQ and P96. The corresponding induction to CYP3A4 was also speculated. Western blotting was employed to detect the changes CYP3A4 protein expression. The possible metabolic mechanism was also explained for understanding the possibility of self-induction or drug-drug interaction. In addition, the genetic polymorphism of PXR might be the important cause leading to the individual differences in CYP3A4 metabolism. Therefore, it is necessary to study the difference in activation effect between PXR wild type and mutants. Docking was also adopted to simulate PZQ and P96 combined with PXR, which contributed to understand their interaction. The results showed that R-PZQ, S-PZQ and rac-PZQ all had strong activation, and were potent agonists of PXRwt. While R-P96 had moderate activation, rac-P96 had weak activation and S-P96 had no activation. There were some differences in PXRwt stereoselectivity activation between enantiomers and racemate of PZQ or P96. Concentration dependence was found in the activation of PXRwt. Besides, the induction to CYP3A4 protein was tested by Western blotting. Results were in conformity with the reporter gene assay. Docking results showed that PZQ and P96 combined with PXR by hydrogen bonding and hydrophobic effect. PZQ (R-PZQ, S-PZQ and rac-PZQ) and P96 (R-P96, rac-P96) had enzyme induction phenomenon. On the one hand enzyme induction may reduce the effects of drug, resulting in low therapeutic effect. On the other hand, enzyme inhibition may increase the quantity of intermediates and product, leading to occurrence of side effects or toxicity. Hence, this research is of great significance for clinical guideline of PZQ and development of P96.