The Research Of Riccardin D’s Pharmacokinetics,Quality Control And Synthesis Of Its Derivative

1. Research backgroundRiccardin D, a macrocyclic bisbibenzy extracted from Chinese liverworts Dumortiera hirsute, has been shown to have antifungal and anticancer activities both in vitro and in vivo. For antifungal activity, Riccardin D interfered with the biofilm formation of Candida albicans through downregulating the expression of hyphase specific genes and inhibiting the formation of hyphase and exhibited antifungal activity against Candida albicans, and also found to reverse fungal resistance to flueonazole by increasing aecumulation of flueonazole in the Calbieans. For anticancer activity, in vitro, RD exhibited cytotoxic effects against multiple cancer cell lines, such as KB, MCF-7, PC3, and human lung carcinoma cells via multiple mechanisms including anti-tubulin, antiangiogenic effects. Riccardin D had a significant antiproliferative effect on human leukemia cell lines HL-60, but showed no effect on the topoisomerase-Ⅱ-deficient HL-60/MX2cells. The pBR322DNA relaxation assay revealed that riccardin D selectively inhibited the activity of topoisomerase Ⅱ and the susppression of topoisomerase Ⅱ activity by riccardin D was stronger than that of etoposide, a known topoisomerase Ⅱ inhibitor. In vivo, RD exhibited significant inhibitory effects on the growth of human NSCLC tumors in a xenograft mouse model and APCmin/+model, the inhibitory effects by riccardin D was similar to etoposide but without obvious toxicity to animals after three weeks injection. Although accumulating evidence demonstrate that riccardin D is a novel NDA topoisomerase Ⅱ inhibitor and considered as a potential candidate compound for treatment of cancers, the pharmacokinetic studies, a critical component in drug development process, are missing. Herein, we investigated the absorption, metabolism, tissue distribution and excretion of RD in rats to provide valuable pharmacokinetic information to facilitate the development of RD as an anticancer drug.2. Synthesis of riccardin D’s metaboliteThe metabolite of riccardin D was synthesized and the structure was confirmed by HPLC\MS via compared with that in bile samples.3. The absorption,distribution,metabolism,and excretion of Riccardin D in ratsThe present study was designed to investigate the pharmacokinetics following oral and intravenous administration of Riccardin D, an anticancer drug candidate isolated from Chinese liverworts Dumortiera hirsute, in Wistar rats. An HPLC-MS/MS analytical method was developed and validated. The results demonstrated that Riccardin D’s bioavailability was13.4%,11.4%, and9.8%after oral administration at20mg/Kg,40mg/Kg, and80mg/Kg, respectively. There was no significant difference in the elimination half-time of Riccardin D at these doses, suggesting that Riccardin D may have linear pharmacokinetic characteristics in rats. The metabolite of Riccardin D in rat was identified as the glucuronide of Riccardin D. Riccardin D showed a wide distribution in various tissues followed by a rapid elimination from most of the tissues tested. Riccardin D was found to distribute widely in the tissues0.5h after oral and intravenous administration. The tissue concentrations were markedly decreased8h and6h after oral and intravenous dosing, respectively. Both Riccardin D and its conjugated metabolite were detected in urine and bile samples while only Riccardin D was detected in feces. The metabolite and metabolic pathway of riccardin D in vivo was studied. In vivo, rat biles were collected after administration of riccardin D and were analysized by HPLC-MSn method after disposition. the metabolite of riccardin D in rat was identified as the glucuronide conjugate of riccardin D for the first time. Taken together, the study provided valuable pharmacokinetic data for further drug development of Riccardin D.4. P450inhibition activityCYPs(CYP1A2,CYP2C19,CYP3A4) high throughput inhibitor screening kit were used and the methods were validated by their positive control inhibitors. The IC50was over20μM to CYP1A2,2.83μM to CYP2C19and1.70μM to CYP3A4.5. The study on the riccardin D’s quality control The method of riccardin D’s quality control was studied and the standard was established.6. Conclusion and prospectThe HPLC-MS/MS method was simple, rapid and sensitive enough to meet the requirements for the quantitation of riccardin D in biology samples.Riccardin had a low oral bioavailability, which limits its development. The study to prepare the nanocrystals to enhance its dissolution is undergoing.Riccardin D shows a wide distribution in various tissues followed by a rapid elimination from most of these tissues. After intravenous administration, Riccardin D had a higher content in lung. Therefore it has potential to be a lung cancer inhibitor. This is in accordance with the previous study showing that RD had promising inhibitory effect on human lung cancer cells in vitro and in NSCLCH460xenografts bearing mouse mode. The results of metabolism study in vivo demonstrated that riccardin D’s metabolite was the conjugate with glucuronate of phase II metabolism and was identified as a mixture of two enantiomeric atropisomers. The synthesis of the conjugate is undergoing and the synthesized conjugate will be useful in further study on riccardin D preparation’s pharmacokinetics. Riccardin D has no inhibitory action to CYP1A2but has inhibitory action to CYP2C19and CYP3A4. The established standard can be used for the quality control of riccardin D.