Assessment Of The Role Of Drug Transporters In The Pharmacokinetics And Renal Excretion Of Ginkgolides And Bilobalide

Ginkgo biloba L is an ancient Chinese phytomedicine,which has been valued for many centuries for its medicinal properties.The use of Ginkgo biloba as dietary supplements has increased dramatically,making Gingko biloba one of the top-selling phytopharmaceuticals.The ginkgo biloba extract(GBE)contain many bioactive compounds,with terpene lactones and flavanol glycosides being the major active components.Terpene lactones(ginkgolides A,B,C,and bilobalide)are said to occur exclusively in Ginkgo biloba L.constitute 5-7%of the total bioactive components.Terpene lactones have been used for the protection of cardiovascular,cerebrovascular,and neurodegenerative diseases such as dementia and Alzheimer's disease.It is a potent antioxidant and antagonizes platelet activating factor(PAF).However,knowledge of the biological fate,including disposition pathways and kinetics in the body should be explored to promote optimal use of such phytomedicine.Previous investigations have shown that the bioavailability of ginkgolides and bilobalide is high,and following oral administration,significant amounts were reported to be excreted unchanged in urine.Tissue concentration declines by several-fold during the first 6 h and the metabolites are mainly excreted in the urine,feces,and traces by the bile.These biological processes suggest the involvement of drug metabolizing enzymes and transporters in the absorption,distribution,metabolism and excretion(ADME)of these bioactive compounds.Ginkgolides are lipophilic,are easily ionizable at physiologic pH rendering them potential uptake or efflux substrate of drug transporters.Moreover,information on the transporters involved in the pharmacokinetics and fate of ginkgolides and bilobalide is limited.The overall objective of this study was to investigate the interaction of both drug uptake and efflux transporters expressed at different tissues especially the kidney and the brain,and provide insight into the role that these transporters play in impacting on the ADME of ginkgolides and bilobalide in the living system.1.Development and Validation of UPLC-MS/MS Method for the Determination of Ginkgolides and Bilobalide in BiosamplesWe developed and validated an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for quantitation of ginkgolide A,B,C,and bilobalide simultaneously in plasma,tissues,and urine as well as in different cell lines expressing transporter proteins.The assay involved an optimized simple sample handling with methyl tert-butyl ether for liquid-liquid extraction and reconstitution in modified dissolution solution.Pretreatment of samples with 50 ?M ascorbic acid and the addition of ascorbic acid and formic acid in dissolution solution significantly reduced matrix effect and stabilized the post-preparative samples.Separations were performed by Zobrax RRHD column(extend-C18 1.8?m,3.0 x 100mm)with acetonitrile gradient elution(eluent A:0.1%formic acid in water,eluent B:0.1%formic acid in acetonitrile).The analysis was carried out in the negative ion scan mode using multiple reaction monitoring.For the determination of ginkgolides and bilobalide in the rat plasma,tissues,and urine,no cross-interference among the analytes and blank was observed.The method was validated for linearity at a concentration range of 4-1000 ng/mL,LLOQ was 4 ng/mL,accuracy(85.0%-114.9%),precision(<7.32%),recovery(77.38%-107.4%),except ginkgolide C,matrix effect(92.1%-116.0%)at three QC concentration levels.The compounds were stable under the most experimental conditions,so ginkgolides and bilobalide should be analyzed immediately after samples were prepared.For the determination of ginkgolides and bilobalide in the cell lysate,no cross-interference among the analytes and blank was observed.The method was validated for linearity at a concentration range of 20-5000nM,LLOQ was 20 nM,accuracy(88.3-114.9%),precision(<11.0%),recovery(94.31-105.9%),matrix effect(93.8-111.0%)at three QC concentration levels.The compounds were stable under the experimental conditions2.Pharmacokinetics and Tissue Distribution Study of Ginkgolides and Bilobalide.By using the UPLC-MS/MS analytical method,we carried out pharmacokinetics study,tissue distribution study,and comparative pharmacokinetics study of pure bilobalide and bilobalide in the extract in rats.Results of the pharmacokinetics study showed that ginkgolides and bilobalide are rapidly absorbed into the systemic circulation reaching peak plasma concentration at about 1.5 h for ginkgolide A,B and bilobalide.This rapid absorption phase was followed by a prolonged elimination phase in rat's plasma indicating a bi-phasic fate in their elimination.Ginkgolide C resulted in low concentrations,and this is attributed to its extensive metabolism in the blood.Ginkgolides A,B and bilobalide showed good oral bioavailability and are widely distributed in tissues with liver and kidney showing high accumulation while tissue like the brain showed minimal accumulation.The comparative study between pure bilobalide and bilobalide in the extract indicated that when give as extract,bilobalide is more abundant in systemic circulation as the Cmax,AUC,and bioavailability was significantly higher than the group that was administered pure bilobalide.3.Influence of OAT 1/3 on the Pharmacokinetics and Disposition of Ginkgilides and BilobalideTerpene lactones are predominantly eliminated via the renal pathway,and we hypothesize that renal transporter,organic anion transporter 1(OAT 1),and organic anion transporter 3(OAT3)may be involved in the renal elimination of ginkgolides and bilobalide.To explore this hypothesis,studies on pharmacokinetics,kidney accumulation and urinary excretion of ginkgolides and bilobalide in rats were carried out.Also,uptake of these compounds in MDCK and human embryonic kidney 293(HEK293)cells overexpressing OAT1 or OAT3,respectively was studied.In vitro data demonstrated that both OAT1 and OAT3 are involved in the active transport of these terpene lactones.Uptake of ginkgolide A,B and bilobalide by MDCK-OAT1 cells showed significant differences compared to the uptake by MDCK-mock cells.indicating the involvement of OAT1 in the transport of ginkgolide A,B and bilobalide.The uptake by MDCK-OAT1 was 3-,4-,and 3-fold higher than the MDCK-mock for ginkgolide A,B and bilobalide,respectively.This observed difference indicated that ginkgolide A,B and bilobalide are likely substrates of OAT1.Furthermore,ginkgolides and bilobalide uptake by MDCK-OAT1 cells were significantly reduced or inhibited when it was incubated with 1 mM probenecid.This further confirmed that ginkgolide A,B and bilobalide are substrates of OAT1.In the same vein,the uptake of ginkgolide A,B and bilobalide into HEK293-OAT3 was observed to be 5-7-fold higher than the HEK293-mock cells which also is indicative of active uptake of these compounds by OAT3.Also,in the presence of probenecid OAT3-mediated uptake was significantly reduced.OAT 1-and OAT3-mediated ginkgolide A,B and bilobalide transport was further characterized by studying concentration-dependent transport.OAT3 was demonstrated to transport ginkgolide A,B and bilobalide about 2 times faster than OAT1 on the average.Further,the transport of ginkgolide A and B by both OAT1 and 3 appeared to happen over 3-fold faster than the measured rate for bilobalide.In essence,OAT 1/3 appeared to be more efficient at the transport of ginkgolide A and B than bilobalide.Our findings suggest that ginkgolide A,B,and bilobalide are transported by OAT 1/3 in their renal excretion and that these transporters play a role in their PK behavior.Also,in vitro data suggest that OAT 1/3 appeared to be more efficient at the transport of ginkgolide A and B than bilobalide while OAT3 contribute more relative to OAT1 in the renal excretion of ginkgolides.Based on in vitro study,in vivo study was performed.Following inhibition of OAT1/3 activity by probenecid administered at 50 mg/kg 15 min before the administration of ginkgo biloba extract at a dose of 100 mg/kg,the plasma concentrations of ginkgolide A,ginkgolide B,ginkgolide C and bilobalide increased significantly.There was a significant increase in the AUC in the probenecid-treated rat treated(893.48 vs.1123.85?g/L*h,314.91 505.74?g/L*h,and 2724.97 3096.40?g/L*h for ginkgolide A,ginkgolide B and bilobalide,respectively.The renal clearance was observed to be reduced significantly in the probenecid-treated group.In addition,comparison of the PK profile of ginkgolide A,B,C and bilobalide in the kidney showed significant differences in AUC,Cmax and ti/2.The AUC0-t values showed a significant 1.4,3.1 and 1.2-fold decrease for ginkgolide A,B and bilobalide,respectively when given with probenecid.Cmax of the untreated group showed 1.8,2.4 and 1.5-folds increase for ginkgolide A,B and bilobalide,respectively,compared to the treated group.To further characterize the interactions between ginkgolide A,B,C or bilobalide and probenecid,the renal excretion was assessed.Cumulative urinary recovery of these compounds of GBE over 36 h was 22.0,79.0,5.0 and 55.0%for ginkgolide A,B,C and bilobalide,respectively,when administered alone.When GBE and probenecid were co-administered,cumulative urinary excretion decreased to 13.0,59.0,7.0 and 32.0%for ginkgolide A,B,C and bilobalide,respectively.4.In vitro characterization of OCT2-,MATE1-and MATE2K-mediated Transport of Ginkgolides and BilobalideGiven that OAT 1/3 are uptake transporters at the basolateral membrane of renal proximal tubule cells.we hypothesized that the renal tubular transport of ginkgolides and bilobalide might involve multiple membrane carrier systems.We characterized the transport of ginkgolides and bilobalide in MDCK cells overexpressing organic cation transporter 2(OCT2),multidrug and toxin extrusion 1(MATE1 and multidrug and toxin extrusion 2-K(MATE2-K).The intracellular concentrations of ginkgolides A,B,C and bilobalide were determined in Madin-Darby canine kidney(MDCK-mock)and MDCK expressing OCT2,MATE1 and MATE2-K cells in uptake study.Results suggested that OCT2 is involved in the renal disposition of ginkgolide A,B and bilobalide.Ginkgolides and bilobalide showed 40-60%inhibition of 1-methyl-4-phenylpyridinium(MPP+)MATE1-and MATE2-K-mediated uptake at a concentration of 100 ?M.However,the observed inhibitory effect by ginkgolides and bilobalide is considered to have no clinical significance since the reported mean human maximum plasma concentration was in the range of 42-105 nM,22-43 nM,and 58-180 nM tor ginkgolide A,B,and bilobalide,respectively.Ginkgolides and bilobalide are significantly transported by MATE1 and MATE2-K and transport kinetic data showed that ginkgolides and bilobalide are low-affinity substrates to MATE1/2-K with Km values of 5 1 1-2219 ?M.MATE1 intrinsic clearance(CL)values of 1.29 ?L/mg/min and 1.20 ?L/mg/min for ginkgolide A and B,respectively,are higher than that of MATE2-K for ginkgolides A clearance(0.8?L/mg/min),indicating that MATE1 has more efficiency in the transport of ginkgolide A and B.However,both MATE1 and MATE2-K were demonstrated similar affinity to bilobalide with Km of 1476 ?M and 1375 ?M,respectively.Also,their transport efficiency appeared the same(0.3 ?L/mg/min),suggesting a similarity between the two transporters in the uptake of bilobalide as their substrate.In summary,our findings suggest that MATE 1/2-K are involved in the vectorial transport of ginkgolide A,B and bilobalide renal elimination.5.Permeability of Ginkgolides and Bilobalide in HBMECs and MDR1-,BCRP-,MRP2-Overexpressing CellsWe established a BBB model using the human brain microvascular endothelial cells(HBMEC)to study the bidirectional transport of ginkgolides and bilobalide.Interaction of GBE and 0.1 mM verapamil in the transport of ginkgolide A,B,C and bilobalide was also undertaken in the efflux direction.Cellular accumulation of ginkgolide A,B,C and bilobalide in MDR1,BCRP and MRP2 were evaluated,and rat brain accumulation of the compounds was measured following single oral administration of GBE at 100 mg/kg.The results showed the efflux ratios of ginkgolide A,B,C and bilobalide to be in the range of 1.67-2.37 suggesting that the compounds were subjected to some efflux.Also,from the experiment of ginkgolides and bilobalide transport across HBMEC in the BL-AP direction,it was observed that the efflux transport of ginkgolides and bilobalide was significantly inhibited when incubated with verapamil,a potent MDR1 inhibitor.Further,the interaction of ginkgolides and bilobalide with MDR1,BCRP and MRP2 cells in the accumulation study showed significant efflux on ginkgolide A,B,C and bilobalide in MDR1 relative to their respective parental cells which is also inhibited by verapamil.No significant difference was observed between BCRP or MRP2 and their respective parental cells(LLCPK1-mock and MDCK-mock,respectively).This result further confirmed that MDR1 involved in their transport.Permeability measurement both in vitro in HBMECs transmembrane monolayer and in vivo in rats indicated that ginkgolide A,B,C and bilobalide are low permeable compounds.The brain-plasma ratio of concentration of ginkgolide A,B and bilobalide was 1.02,4.01 and 1.28,respectively,at 5 min after oral administration which sharply declined to 0.06,0.11,and 0.08,respectively,at 1 h.Overall,the poor brain bioavailability of ginkgolide A,B,C and bilobalide may be via the mechanism efflux activity of MDR1.6.SummaryA bioanalytical method was developed and validated for in vitro and in vivo assessment of the role of drug transporters in the pharmacokinetics,distribution,and renal excretion of ginkgolides and bilobalide.Results demonstrated that the renal elimination of ginkgolides and bilobalide is via the mechanism of vectorial transport with both uptake and efflux transporters playing a role.The first step of this process involves uptake of ginkgolides and bilobalide from the blood into the proximal tubular cells by OCT2,OAT1 and OAT3 locate at the basolateral membrane of the tubular cells.Consequently,in the second step,the efflux transporters MATE1 and MATE2-K,as well as MDR1,play the role of mediating the transport from the proximal tubular cells into the urine.Data from the study further enumerated the influence of OAT 1/3 in the pharmacokinetics of ginkgolide A,B,C and bilobalide in the rats.On the other hand,our finding also showed that ginkgolides and bilobalide are poorly bioavailable in the brain and MDR1 could be responsible for its low accumulation in the brain.