The Effect Of Xiao Chaihu Decoction On The Metabolism Of Irinotecan And Its Intestinal Protection

Objective:Irinotecan?CPT-11?is the first-line chemotherapy drug for treating colorectal cancer,but the incidence of diarrhea and other toxic side effects is up to 80%,of which 59%of patients were forced to lower the dosage of drugs,replace chemotherapy drugs even terminate chemotherapy due to severe diarrhea,which is caused by many factors,and seriously affected the effect of therapy and restricted the clinical application.Our previous studies have shown that on the premise of not affecting the anti-tumor effect of CPT-11,xiaochaihu decoction?XD?could reduce the blood stool rate of mice with delayed diarrhea and improve the intestinal tissue injury.The objective of this study were?1?to establish a sensitive,rapid and stable UPLC-MS/MS method for the determination of CPT-11 and its metabolites in vivo also the 9 active ingredients in XD,in order to provide a reliable detection method for the subsequent studies;?2?using rat in-situ intestinal perfusion model to examine XD influence the metabolic process in vivo.By measuring CPT-11 and its metabolic products and content of the main active components in XD,lay a foundation for the study of the mechanism.?3?to explore the mechanism of XD in inhibiting the metabolic process in vivo of CPT-11.And provide new ideas and methods for improving the clinical application of irinotecan and the other anticancer drugs have the similar toxic effect.Method:?1?Establishment of UPLC-MS/MS method for determination of CPT-11and its metabolites in biological sample:A BEH C18 UPLC column was selected as separation column at 40?.The 0.1%formic acid water was used as mobile phase A and0.1%formic acid in acetonitrile was used as mobile phase B.The flow rate was 0.4 ml/min and the injection volume was 5?L.A triple quadrupole mass spectrometer equipped with an electrospray ionization?ESI?source was used for mass spectrometric detection.The analytes were quantified using the multiple reaction monitoring?MRM?mode and positive ion scan mode.m/z 587.37?124.1 for CPT-11,m/z 393.17?249.09 for SN-38,m/z569.27?393.17 for SN-38G,m/z 349.21?219.04 for CPT?I.S.?.?2?Establishment of UPLC-MS/MS method for determination of active ingredients in XD in plasma:A BEH C₁₈UPLC column was selected as separation column at 45?.The 0.1%formic acid water was used as mobile phase A and 0.1%formic acid in acetonitrile was used as mobile phase B.The flow rate was 0.4 mL/min and the injection volume was 10?L.A triple quadrupole mass spectrometer equipped with an electrospray ionization?ESI?source was used for mass spectrometric detection.The analytes were quantified using the multiple reaction monitoring?MRM?mode.Wogonin,baicalin,glycyrrhizic acid,saikosaponin d,saikosaponin a,ginsenoside Rg1,ginsenoside Re and daidzein were using positive ion scan mode.While,liquiritin and rutin were using negative ion scan mode.The parameters were m/z 285.16?270.11 for wogonin,m/z 447.24?271.15 for baicalin,m/z 471.50?149.13for glycyrrhizic acid,m/z 803.75?203.16 for saikosaponin d,m/z 803.75?331.21 for saikosaponin a,m/z 23.72?643.59 for ginsenoside Rg1,m/z 969.77?203.1 for ginsenoside Re,m/z 417.29?135.00 for liquiritin,m/z 609.44?300.23 for rutin,m/z255.09?199.16 for I.S.?daidzein?.?3?Investigating the effect of XD influence the metabolic process of CPT-11 in vivo by using the in-situ intestinal perfusion model:12male SD rats were randomly divided into control group and experimental group for the experiment of intestinal perfusion in vivo.The bile,urine,intestinal perfusion liquid and tail tip blood were collected at the 0h,0.5h,1h,1.5h,2h and 2.5h after injection of CPT-11by microdosage syringe pump via jugular vein.Analyte in biological samples was extracted with protein precipitation agent.CPT-11 and its metabolites in plasma,bile,urine,intestinal perfusion liquid and active ingredient concentration of XD in plasma were detected with established UPLC-MS/MS method.?4?Mechanism study of XD affect the metabolic process of CPT-11:120 healthy male C57BL6 mice were randomly divided into 6 groups after the adaptability of feeding high fat forage three weeks,each group 20:Blank control group?equal volume of physiological saline?,Model control group?equal volume of physiological saline?,XD high dose group(3000 mg·kg^-1·d^-1),XD middle dose group(1500 mg·kg^-1·d^-1),XD low dose group(500 mg·kg^-1·d^-1),Loperamide group(0.48mg·kg^-1·d^-1).All mice were gavage one time a day for 15 days.On day 4 to 8 of the experiment,all mice except the blank control group were intraperitoneally injected with CPT-11?75 mg·kg-1·d-1?to induce delayed diarrhea.All mice put to death with ether anesthesia at 16th day.The duodenum,jejunum,ileum,colon tissues were collected and homogenated.ELISA kits were used to measure biochemical indexes,including TNF-?,COX-2,PGE₂,IL-1?,IL-1?,IL-6,IL-10,IL-15.In order to explore the mechanism of XD in antagonizing the intestinal toxicity of CPT-11.Result:?1?Establishment of UPLC-MS/MS method for determination of CPT-11 and its metabolites in biological sample:It showed good linear relationship for CPT-11,SN-38and SN-38G in the blank plasma,bile,urine and intestinal perfusion matrix within the range of 0.2673²196.7581 ng/mL?R>0.99?,0.4678¹799.1677 ng/mL?R>0.99?,0.1053²33.3822 ng/mL?R>0.99?respectively.Inter-day and intra-day precision of three different levels range from 0.17%to 2.89%.Accuracy range from-5.42%to 9.30%.This experiment has no matrix effect.The sample is very stable under short-term and long-term conditions.?2?Establishment of UPLC-MS/MS method for determination of active ingredients in XD in plasma:It showed good linear relationship for 9 active ingredients of XD within the range of linear concentration range?R>0.9?.Inter-day and intra-day precision of three different levels range from 0.09%to 14.13%.Accuracy range from-8.79%to 9.92%.The sample is very stable under short-term and long-term conditions without matrix effect.?3?Investigating the effect of XD influence the metabolic process of CPT-11 in vivo by using the in-situ intestinal perfusion model:A.Result of content determination of CPT-11 and its metabolites in biological sample:CPT-11 content in bile,urine,intestinal perfusion fluid was far higher than SN-38 and SN-38G.SN-38concentration in plasma was significantly higher than the CPT-11 and SN-38G.?1?Bile:CPT-11,SN-38 and SN-38G concentrations of the experimental group are higher than the control group in bile.?CPT-11:The control group first increased slowly,then rapidly increased after 120 minutes.The experimental group have been increased sharply,then a slowly increasing trend after 90 minutes.?SN-38:The control group was in a trend of increase at a constant speed.The experimental group was a rising speed decreasing trend before 120 minutes,a rapid decline after 120 minutes,but its concentration was still higher than the control group.?SN-38G:The control group was a trend of increase at a constant speed.The experimental group was first rapid increase then slow increase before the 120 minutes,concentration was reduced rapidly after 120 minutes,and the concentration was lower than the control group.?2?Plasma:There was no significant difference of quantity of CPT-11 and its metabolic products between control group and experimental group in vivo:?CPT-11:The control group peak of drug concentration in blood was at 60 minutes after the treatment,while the experimental group peaked at 120minutes.?SN-38:The control group peak of drug concentration in blood was at 120minutes after the treatment,while the experimental group peaked at 150 minutes.?SN-38G:The control group peak of drug concentration in blood was at 60 minutes after the treatment,while the experimental group peaked at 30 minutes.?3?Intestinal perfusion liquid:?CPT-11:The experimental group and control group were a trend of increase.But the concentration of experimental group was significantly higher than control group.?SN-38:The control group showed a trend of increase after the first reduce.?SN-38G:The control group peak of drug concentration in blood was range 90 minutes to 120minutes,while the experimental group was range 120 minutes to 150 minutes.Before 120minutes,concentration of experimental group was lower than the control group.?4?Urine:?CPT-11:Before 120 minutes,the excretion of CPT-11 was slowly rising in both experimental group and control group.After 120 minutes,the control group rose slowly,while the experimental group fell sharply.?SN-38:The experimental group and the control group were increased after the first reduce the trend,but the concentration of experimental group is lower than the control group.?SN-38G:The concentration of experimental group was significantly less than the control group.B.Result of content determination of 9 kinds of active ingredients in XD in plasma:the contents of glycyrrhizic acid,baicalin and wogonin were obviously higher than other ingredients.glycyrrhizic acid,saikosaponin d,saikosaponin a,rutin and ginsenoside Re have similar trends and all concentration peaked at 90 minutes.Concentration of ginsenoside Rg1 and liquiritin were very stable and no obvious changes in vivo.?4?Mechanism study of XD affect the metabolic process of CPT-11:?1?Compared with model control group,high,medium and low dose XD and loperamide could effectively reduce the CPT-11 induction generated content of TNF-?,and medium dose XD had the best effect.?2?High,medium and low dose XD and loperamide could effectively reduce the concentration of COX-2 in the jejunum,and medium dose XD had the best effect.?3?As a result,the high dose XD had the best effect of significantly reducing the concentration of PGE₂ in each intestine segment,and loperamide was the worst.?4?The IL-1?was not significantly changed in the intestinal tracts of mice in different groups.?5?High,medium and low dose XD could lower the concentration of IL-1?to the normal level,while medium and high doses are better.?6?In addition to low doses of XD could cause IL-6 concentration of jejunum higher than the model control group,XD and loperamide could effectively reduce the concentration of IL-6 in gut.Of these high dose XD effect is best.?7?There was no significant difference in the secretion effect of IL-10 in each intestinal segment.?8?From the perspective of promoting the secretion of anti-inflammatory factor IL-15,the effect order is:medium dose XD group>loperamide group>high dose XD group>low dose XD group.Conclusion:?1?Established UPLC-MS/MS method was fast,high sensitivity and good reproducibility.It can be used for determination of CPT-11 and its metabolites in plasma,bile,urine and intestinal perfusion fluid.And it can be used for determining concentration of the active ingredient of XD in plasma.Lay the foundation for research on CPT-11 and XD in vivo.?2?Investigating the effect of XD influence the metabolic process of CPT-11 in vivo by using the in-situ intestinal perfusion model:XD can reduce the diarrhea caused by CPT-11 by inhibiting the bile and intestinal excretion of toxic compounds such as CPT-11,SN-38 and SN-38G.?1?XD start giving play to the role of inhibiting enterohepatic circulation process of CPT-11 after 90 minutes and SN-38 and SN-38G after 120 minutes.Therefore,the contents were decrease from lumen to the liver.?2?The concentrations of three compounds,including CPT-11,SN-38 and SN-38G,were not affected by XD in plasma.This indicates that XD will not affect the anticancer effect of CPT-11.?3?CPT-11 mainly in the form of original medicine via bile excreted to lumen.XD could promote CPT-11 and inhibite SN-38 efflux via bile.Within 120 min,XD could inhibite SN-38G excretion,while after 120 min,XD promote SN-38G excretion.?4?CPT-11 mainly in the form of original medicine via urine excreted.XD can effectively inhibit the excretion of CPT-11,SN-38 and SN-38G in urine.?5?The most important active components of XD may be glycyrrhizic acid,baicalin and wogonin.