Clinical Studies Of The Drug Interaction Between Compound Herbal Preparation Of Sailuotong And Atorvastatin And Analysis Of The Related Mechanism

With the increasing development of science and technology and Chinese medicine,a unique medical pattern of combining Chinese and Western medicine has been formed in China.A more and more common use of the combination inevitably leads to some clinical problems,namely drug interactions between Chinese and Western medicines.Sailuotong(SLT)is a standardized herbal medicine formula made from extracts of ginseng(the dried root and rhizome of Panax ginseng C.A.Meyer),ginkgo(the leaves of Ginkgo biloba L.),and saffron(the stigma of Crocus sativus L.),and is used to treat vascular dementia(VD)with Qi deficiency and blood stasis.Since statin is a common base drug for VD patients combining the two is highly probable.To ensure the efficacy and safety of the combined treatment,this project aimed to investigate the potential clinical drug interaction between compound herbal preparation of SLT and atorvastatin(AT)through clinical studies,and analyze the related mechanism afterwards by using in vitro models.Part I.Clinical study of the interaction between SLT and AT1.Establishment and validation of the bioassay methodIn this study,a rapid and sensitive LC-MS/MS assay for AT and its metabolites ohydroxy atorvastatin(2-HAT),p-hydroxy atorvastatin acid(4-HAT),atorvastatin lactone(ATL),o-hydroxy atorvastatin lactone(2-HATL)and p-hydroxy atorvastatin lactone(4HATL)was established.Plasma samples were acidified and processed by protein precipitation method.Gradient elution was performed with methanol:acetonitrile(1:1)as the organic phase and water:methanol:acetonitrile(9:0.5:0.5)as the aqueous phase,both containing 0.05%formic acid.The electrospray ionization source was scanned in positive ion mode,with multiple reaction monitoring.Relative quantification was used for 2-HATL and 4-HATL.The results showed that the pretreatment method effectively prevented the conformational transformation between acid and lactone forms.AT and its metabolites showed good linearity in the range of 0.1～25 nmol·L-1.All components to be tested are measured simultaneously in 5.5 min.RSD of precision and RE of accuracy were less than 15%.The stability was good under various conditions.The method established for the simultaneous quantification of AT and five metabolites in human plasma is accurate,rapid,sensitive and stable,which can be used for clinical pharmacokinetic studies.2.Drug interaction study between SLT and AT in healthy subjectsTo investigate the interaction between SLT and AT in the clinical setting,a study of the interaction between the herbal compound SLT and AT in healthy subjects was first conducted.Ten healthy Han Chinese males with normal liver and kidney function were screened and enrolled in the study,which was designed as an open-label,single-sequence,self-controlled,two-period study.The pharmacokinetic effects of single(120 mg)and continuous(120 mg,bid)doses of SLT on AT(20 mg)were investigated separately.Plasma concentrations of AT and each metabolite were measured by LC-MS/MS method and pharmacokinetic parameters were calculated.Safety and lipid levels were assessed by laboratory tests.The results showed that AUC0-48 and Cmax of AT increased by 12.2%and 37.5%,respectively,after a single dose of SLT compared to the pre-dose period.AUC0-48 and Cmax of AT decreased by 9.7%and 26.3%(P<0.05),respectively,after consecutive doses of SLT.The AUC0-48 and Cmax of AT were significantly decreased by 19.5%and 46.5%,respectively,after consecutive doses of SLT compared to single administration The metabolic rates of the major metabolites were decreased after a single dose of SLT.Laboratory results did not show abnormalities in myotoxicity or liver function indicators.CK was reduced by 30.0%(P<0.01)and TC by 19.6%(P<0.001)compared with preclinical levels.3.Drug interaction study of SLT and AT in VD patientsTo investigate the extent of drug-drug interactions in patients with the combination of the two drugs,a study of SLT and AT in combination in patients with VD was conducted.Thirty-five VD patients with normal liver and renal function and were taking atorvastatin or pitavastatin(PIV)calcium were enrolled.The study followed an open-label,single-sequence,self-controlled design.Venous blood samples were taken at 12 h after statin dosed on the first day of the trial,and a daily dose of SLT(120 mg,bid)was followed therafter.Blood samples at the same time point were taken again after continuous medication of SLT for 1,2 and 3 months.Plasma concentrations were measured by LC-MS/MS to analyze the effect of SLT on the systemic exposure of AT and PIV,and the safety and efficacy-related parameters were monitored during the study.The results showed that the blood concentration of AT and its metabolites decreased after 3 months of continuous combined administration of SLT,with AT,4-HAT,ATL and 4-HATL showing the most significant decreases(P<0.05),and the blood concentration of PIV decreased approximately by 10 folds.Safety results showed no liver function abnormalities or myotoxicity,and no adverse events occurred during the study.TG,TC,LDL and HDL remained within the clinically normal range throughout the study.4.Pharmacokinetic study of SLT and AT in VD patientsThe study was based on a small-sample clinical trial to investigate the effect of SLT on the pharmacokinetic behavior of AT in patients with VD with Qi deficiency and blood stasis in combination.The study followed an open-label,self-controlled,two-period design.Two patients with normal liver and kidney function with Qi stagnation and blood stasis type VD were screened for inclusion,and received AT alone(20 mg,qd)or combined with SLT(180 mg,bid)for 1 8 days in each period.Venous blood was collected from 0 to 24.5 h after AT on the first and last day,and plasma concentrations of AT and each metabolite were measured by LC-MS/MS method and pharmacokinetic parameters were calculated.The results showed that the AUC and Cmax of AT and its five metabolites were substantially reduced by 70%～80%and the half-life was prolonged by 1～3 times after continuous co-administration of SLT compared with AT alone.The above clinical findings suggest that the combination of SLT and AT may cause significant herb-drug interactions,with single and continuous administration of SLT having different effects in healthy subjects.Continuous administration of SLT significantly reduced AT exposure levels in healthy subjects and VD patients.The effect of long-term combined drug administration on metabolic rate could not explain the decrease in AT exposure levels,and it was speculated that the decrease in total bioavailability may be related to the decrease in absorption levels,and the mechanism involved needs further investigation.Part Ⅱ Study on the mechanism of SLT and AT interaction1.Effect of SLT on human intestinal metabolic enzymes and transportersHuman intestinal adenocarcinoma epithelial cells(LS174T cells)were selected as a model for the study of intestinal drug-drug interaction(DDI),and the expression of metabolic enzymes,transporters and their upstream regulatory genes in LS174T cells were detected by RT-PCR.The method for assessing cellular uptake activity using rhodamine 123,AT and PIV as substrates was optimized.The effects of SLT on transporter MDR1,cellular uptake of statin activity and induction of uptake-related metabolic enzymes and transporters and source components were analyzed by adding different concentrations of SLT and Ssffron(XHH),Gingko(YX)and Ginseng(RS)extracts in co-culture.The results of a single co-culture showed that high concentrations of SLT significantly promoted the intracellular uptake of rhodamine 123 and significantly promoted the uptake of AT and PIV by intestinal cells.XHH significantly promoted the intracellular uptake of the three substrates.It was suggested that the inhibitory effect of SLT on MDR1 was mainly due to XHH.48 h incubation results showed that high concentrations of SLT,high concentrations of XHH,YX and RS could significantly increase the intracellular substrate concentrations,while low concentrations of SLT and XHH,YX and RS decreased the intracellular substrate concentrations.RT-PCR results showed that both high concentrations of SLT and XHH could significantly downregulate MDR1 and OATP2B1 transcript levels,and low,medium and high concentrations of XHH and medium and high concentrations of YX could upregulate CPY3A4.2,Effect of SLT on human transporter OATP2B1HEK293T cells were used as an expression line and the HEK293-OATP2B1 cell model was established by transferring the OATP2B1 plasmid into the nucleus by electroporation.After confirming the expression and function of the model and optimizing the activity assay conditions,the uptake of the substrate PIV or AT by the cells was perturbed using the medium containing different concentrations of SLT,XHH,YX and RS total extracts,and the intracellular substrate levels were determined by LC-MS/MS.The results showed that the intracellular substrate concentrations in SLT,XHH,YX and RS high and medium concentration groups were significantly lower than in the control group when AT was used as substrate,and the intracellular substrate concentrations in YX and RS low concentration groups were significantly higher than in the control group.The intracellular substrate concentrations in SLT,XHH medium and high concentration groups and XHH low concentration group were significantly lower than in the control group when PIV was used as the substrate.3.Effect of SLT on human transporter MRP2MDCK cells were used as an expression line and the MRP2 plasmid was transferred into the nucleus by electroporation.After confirmation of the expression and function of the model and optimization of the activity assay conditions,the cellular uptake of the substrate was intervened with different concentrations of SLT,XHH,YX and RS total extracts,and the intracellular fluorescence of the MRP2-specific substrate CMFDA and concentrations of AT and PIV were determined.The results showed that low,medium and high concentrations of SLT and medium and high concentrations of YX and RS increased the intracellular CMFDA concentrations.Low,medium and high concentrations of SLT and medium and high concentrations of XHH,YX and RS increased intracellular AT concentrations.Low,medium and high concentrations of SLT and medium and high concentrations of XHH caused significantly higher intracellular PIV concentrations than the control group.In this part of the study,a clear transporter-mediated mechanism of SLT-AT interaction was investigated in several cellular models.SLT and its components inhibited MDR1,MRP2 and OATP2B1 activity and induced MDR1 and CYP3A4 transcript levels.The increase in statin concentration caused by a single dose of SLT may be due to the inhibition of MDR1 and MRP2 efflux activity,whereas the decrease in statin concentration caused by continuous administration of SLT is associated with the induction of intestinal MDR1 activity levels,the down-regulation of uptake transporter OATP2B1 expression and activity levels,and the upregulation of metabolic enzyme C YP3A4 expression levels.The available results showed that XHH was the main source component of the effect.In summary,this study systematically investigated the herb-drug interactions produced by the combination of SLT and AT in healthy volunteers and clinical patients,and found that the combination produced different effects in single and continuous dosing,with continuous dosing producing a greater reduction in AT plasma exposure levels and a stronger effect in patients.Further mechanistic analysis showed that SLT inhibited the intestinal uptake transporter OATP2B1,the efflux transporter MDR1 and,MRP2,and induced upregulation of MDR1 and CYP3A4 and downregulation of OATP2B1 transcript levels.Based on this complex interaction mechanism,SLT may have different combined effects on absorption at different concentrations and at different sites in the intestine.The results of this study may provide a scientific basis and guidance for the rational clinical use of SLT,and provide a methodological reference for the study of compounded HDI in Chinese medicine.