Study On The Anti-tumor Effect And Pharmacokinetics Of RPDQ

RPDQ is a new ginsenoside semi-synthesized for the first time.In this paper,the semi-synthesis,anti-tumor activity,pharmacodynamics mechanism and pharmacokinetics of RPDQ were systematically studied.1.Semi-synthesis of RPDQTaking PDQ and D-ribose as raw materials,a new ocotillol-type ginsenoside RPDQ characterized with side chain of pentose was semi-synthesized.The yield rate and the purity were 38.7% and 99.0%,respectively.Its structure was characterised as(20S,24S)-12-O-?-D-furan ribosyl-dama-20,24-epoxy-3?,12?,25-triol by HR-MS and NMR.2.Anti-tumor activity of RPDQMolecular docking technique for virtual screening was applied to simulate the binding mode and interaction between RPDQ and tumor-related proteins.As a result,22 possible target proteins of RPDQ were screened out.The proteins with higher function scoring were chosen by related literatures,then the inhibition effect on proliferation of RPDQ were tested in vitro by MTT assay on S180 cells,human lung adenocarcinoma SPC-A-1 cells and A549 cells.The results showed that RPDQ could inhibit the proliferation of above three cancer cells in a concentration-dependent manner,of which S180 cells suffered the most.The study on the effect of RPDQ in S180 tumor-bearing mice showed that: compared with model group,RPDQ at dose of 36 mg/kg and 18 mg/kg could significantly reduce the weight of solid tumors(P<0.01 and P<0.05).The inhibition rates were 22.50% and 14.25%,respectively.Meanwhile,the spleen index(P<0.01,P <0.05)and thymus index(P<0.01,P<0.05)of the high and medium dose groups also significantly decreased.Histopathological examination presented disordered arrangement and different sizes in model group,which were obviously difference from normal group.Compared with the model group,the high dose RPDQ group(36 mg/kg)showed significant improvement.In a word,studies in vivo have indicated that RPDQ could inhibit the growth of solid tumor in tumor-bearing mice in a dose-dependent manner,possibly related with immunosuppression.The study on the effects of RPDQ,which was intragastric administrated to mice bearing S180 ascites tumors,showed that: the high dose group prolonged the survival time of ascites tumor-bearing mice for more than 50 days,similar to that of the positive drug cyclophosphamide,which suggested that RPDQ could inhibit S180 ascites tumors in mice.3.Metabolomics Analysis of anti-tumor effect of RPDQ in S180 Tumorbearing MiceBased on UPLC-QTOF-MS and multivariate statistical analysis,the serum samples of S180 tumor-bearing mice treated with RPDQ 36 mg/kg were determined.Compared with normal control group and model group,RPDQ could regulate the contents of 16 potential metabolites such as leukotriene A4,phosphatidylethanolamine,retinyl ester,L-glutamine,indoleacetaldehyde and linoleic acid,which suggested that arachidonic acid metabolism,linoleic acid metabolism,alanine,aspartate and glutamate metabolism,pentose and glucuronate interconversions,retinol metabolism,tryptophan metabolism and glycerophospholipid metabolism were involved in the anti-tumor effect of RPDQ.4.Pharmacokinetics study of rats oral administered RPDQThe absorption,distribution,excretion and metabolism of RPDQ in rats were studied by using UPLC-MS/MS and UPLC-QTOF-MS methods.A UPLC-MS/MS quantitative method for the determination of RPDQ in rat plasma was established.LLOQ,linear range,specificity,accuracy,precision,dilution reliability,extraction recovery,matrix effects,and stability were all meet the requirements of pharmacokinetic studies.The concentration-time profiles of 6,12,24 mg/kg RPDQ administered intragastrically and 0.3 mg/kg RPDQ administrated intravenously showed that RPDQ was slowly absorbed after intragastric administration,the Tmax was 7.25 h,then it was gradually eliminated due to being detected in plasma even 60 h after administration.The absolute bioavailability of RPDQ administered intragastrically was 5.15%.(2)A UPLC-MS/MS quantitative method for the determination of RPDQ in rat tissues was established,and the distribution of 12 mg/kg RPDQ in rats was illustrated.The results indicated that RPDQ was rapidly distributed in stomach,large intestine,small intestine and other digestive tissues.Uterus and liver were also target organs while testis and adipose were less distributed.(3)A UPLC-MS/MS quantitative method for the determination of RPDQ in feces,urine and bile of rats was established,and the excretion of 12 mg/kg RPDQ by intragastric administration in rats was determined.The results showed that excretion rates of RPDQ in feces and urine reached the maximum during the period of 8-12 h and in bile at 6-8h.And cumulative excretion amount within 72 h of RPDQ in feces and urine were 38.30±3.66% and 6.39±0.12 %,respectively.While the cumulative excretion of bile within 36 h was only 0.09%.The total cumulative excretion was 44.69%,which suggested that RPDQ could be eliminated in vivo.(4)Using UPLC-QTOF-MS combined with UNIFI metabolite analysis platform,the metabolites of RPDQ in rats were identified,which indicated that RPDQ was extensively metabolized.A total of 14 metabolites,including 4 phase I metabolites and 9 phase II metabolites,were characterized.Phase I metabolic pathway included dehydration,hydrogenation and deglycosylation.While Phase II metabolic pathways,the predominated pathway,included methylation,hydroxylation,acetylation,sulfation,phosphorylation,cysteine binding,glycine binding,glutathione binding and glucuronidation.In summary,in this study,a new ocotillol-type ginsenoside RPDQ characterized with pentose side chain was semi-synthesized.Pharmacological studies have proved that RPDQ has good anti-tumor effect.On the one hand,it may play an important role on anti-tumor effect through immunosuppression,on the other hand,it may exert anti-tumor effect through regulating seven endogenous metabolic pathways including arachidonic acid metabolism,linoleic acid metabolism,alanine,aspartate and glutamate metabolism,pentose and glucuronate interconversions,retinol metabolism,tryptophan metabolism and glycerophospholipid metabolism.Pharmacokinetic studies suggested that RPDQ was characterized with slow absorption,slow elimination,extensive distribution and metabolism,and good bioavailability.This thesis will provide the theoretical basis and data support for the further research and development of RPDQ.Meanwhile,it could offer a reference of new ideas and approaches to the PK-PD research of the similar drug.Innovations:1.A new ginsenoside RPDQ with D-ribose chain was synthesized for the first time,given the absence of pentoside ginsenosides in nature.2.The anti-tumor activity screening for RPDQ was implemented in vivo and in vitro for the first time,and its anti-tumor effect was confirmed.3.The mechanism of anti-tumor effect of RPDQ was explored by using metabolomics method for the first time and several potent metabolic pathways were identified.4.The pharmacokinetics of RPDQ was studied for the first time,and the main ADME pharmacokinetic parameters were obtained.