| BackgroudIrinotecan(CPT-11),which is the first line therapy agent for the treatment of metastatic cancer of the colon or rectum,has shown promising antitumor efficacy in clinical practice.It is also widely used to treatment of small cell lung cancer,bladder cancer,brain tumor,breast cancer,gastric cancer and other solid tumors.Over 600,000 of patients with colorectal cancer might be treated with CPT-11 every year.It's estimated that up to 80%of patients will experience CPT-11-induced diarrhea(incidence of National Cancer Institute grade 3 or 4 diarrhea is 15-40%).Therefore,dose limiting diarrhea is a major challenge for this widely used chemotherapeutic drug.The mechanisms of CPT-11 induced diarrhea(CID)remains unknown,but it is generally believed that diarrhea is primarily related to intraluminal concentrations of SN38.The intraluminal exposure of SN38 is major regulated by the the efflux transporters and metabilic enzymes.Until now,various strategies have been used to reduce toxicity by efflux transporters and metabilic enzymes-targeted therapy.However,the effects of CPT-11 on the related metabolic enzymes and efflux transporters function,and the relationship between metabolic enzymes and the efflux transporters and the toxicity is still under research.Due to the complicate mechanism,the efficacy of these strategies had limit effect on CID.Traditional Chinese Medicine Xiao Chai Hu Tang(XCHT,Sho saiko to in Japan),as chronicled in Shang Han Lun,is composed of Bupleuri Radix,Scutellariae Radix,ginseng Radix,Glycyrrhizae Radix,Pinelliae Tuber,Zingiberis Rhizoma and Jujubae Fructus.In modern clinical practice,it was widely used to protect on gastrointestinal mucosa and liver cells during CPT-11 chemotherapy in Asian.CID is characterized by watery stool,emesis,diaphoresis,abdominal cramping,and bloody diarrhea.The diarrhea symptoms were consistent with the "Xiali","Xiexie" induced by the weakness of the spleen and stomach in theory of Traditional Chinese Medicine.Treatment of CID with XCHT accorded to the theory of Traditional Chinese Medicine.Furthermore,the protection effects of XCHT on gastrointestinal in chemotherapy was stronger than its derivative prescription TJ-14 and PHY906,which improved the CID.Therefore,we used the XCHT to study the mechanism of its reduced the CID.Objectives1 Effect of XCHT and its components co-administration with CPT-11 on the antitumor activity and toxicity of CPT-11,which were evaluated using nude mice bearing colon cancer.2 To characterise the role of phase ?,? and ? drug metabolism/transport impairment in toxicity and investigate the effects of co-administered herbal formula Xiao Chai Hu Tang(XCHT)on the toxicities and efficacy and the underlying mechanisms.3 Effect of P-glycoprotein(P-gp),Breast Cancer Resistance Protein(BCRP),and Mutidrug Resistance Protein 2(MRP2)on the CID.Methods1 Analysis of main components in XCHT by UHPLC-MS/MS.2 The effects of combination XCHT with CPT-11 on antitumor activity and intestinal toxicity of CPT-11 were evaluated using nude mice bearing colon cancer2.1 Effect of XCHT co-administration on the antitumor activity and toxicity of CPT-11The effects of XCHT pretreatments on antitumor activity and toxicity of CPT-11 were evaluated using nude mice bearing colon cancer.The HT 29 cells were cultured in DMEM medium supplemented with 10%(v/v)fetal bovine serum.The HT-29 cells(5×105 cells/0.1 ml)were inoculated subcutaneously into the right flank of mice.When the mean estimated tumor volume reached about 150~300 mm3 on day 10~14 after tumor transplanted,the mice were randomly divided into six groups(10 mice per group):the vehicle group(i.p saline and p.o.saline),CPT-11 model group(i.p CPT-11 and p.o.saline),and co-administration with different dose XCHT group(i.p CPT-11 and p.o.XCHT).XCHT(3.6 g/kg p.o.high dose,0.9 g/kg p.o.middle dose and 0.225 g/kg p.o.low dose)or saline were orally administered daily 30 min before CPT-11 administration from the day before yesterday(day-2)to 6 days after the start of CPT-11 injection.CPT-11(50 mg/kg i.p.)was consecutively given for 6 days.Compounds group(i.p CPT-11 and pretreated with mixture compounds(baicalin 180 mg/kg:wogonin 54 mg/kg:SSd 36 mg/kg:liquiritin 36 mg/kg:zingerone 36 mg/kg).All mice were monitored the intestinal toxicity index(ITI)from day 6 to day 9 and sacrificed on day 9 of the experiment.Blood was collected from the orbital sinus of anesthetized mouse on day 9.Tissue samples from the distal small intestine,colon and liver of all mice were fixed immediately in 4%polyformaldehyde for 48~72h and embedded in paraffin.2.2 Levels of inflammation in mice with or without XCHT and its components treatment were examined by detecting TNF-?,IL-1?,IL-6,IL-2 and IL-10 in intestinal tissue and plasma,using enzyme-linked immunosorbent assay(ELISA)kits.2.3 Phytochemical Screening and antidiarrhea evaluation of XCHT in mice.In the phytochemical Screening and anti-diarrhea evaluation of XCHT's components study,ten FVB mice of each group was oral administration with baicalin,wogonin,zingerone(ZG),the mixture of saikosaponin d(ssd),liquiritin and ZG,the mixture of wogonin and ZG.Mice were monitored using the ITI and sacrificed on day 9 of the experiment.3.The effect of XCHT co-administration with CPT-11 on protein expression of Cyps and Ugts in liver and intestinal tissue of mice by UHPLC-MS/MS method.Fifty FVB mice were divided into two groups:CPT-11 alone and XCHT combination with CPT-11.Mice were administered with CPT-11(50 mg/kg,i.p)for 6 consecutively days.Body weight,stool consistency and occult blood in stool were monitored daily.In the XCHT co-administration group(i.p.CPT-11,pretreated with XCHT),XCHT(3.6 g/kg p.o.)were given daily starting 2 days before CPT-11 and then daily with CPT-11 administration(30 min before)for 6 days.Five mice from each group were sacrificed on day 0,2,4,6,9 to examined Ugts regulation in response to XCHT and CPT-11 treatments.3.1 Time course study of Cyp and Ugt enzymes expression in mice was monitored by LC-MS/MS at days 0,2,4,6,and 9 post administration CPT-11 alone or XCHT combination with CPT-11.3.2 The effect of XCHT co-administration with CPT-11 on the activity of Ugt enzymes were measured using probe substrate.3.3 Effect of XCHT on the tissue concentration of CPT-11,SN38,and SN38-G at 15min,1h,6h and 24h after the 6 consecutively i.p.administration of CPT-1150mg/kg.4 The effect of XCHT and wogonin:ZG on the UGT1A1 expression in Caco-2 cells.5 Western blot analyses of efflux transporters expression after CPT-11 treatment.6 The effect of efflux transorters on the CPT-11 induced toxicity.Mdrla(-/-)?Mrp2(-/-)?Bcrp1(-/-)and FVB mice were administered with CPT-11(50 mg/kg,i.p)for 6 consecutively days.ITI were monitored daily from day 6 to day 9.Five mice from each group were sacrificed on day 0,2,4,6,9 to examined Ugts regulation in response to CPT-11 treatments.The enzymes expression levels in mice was monitored by LC-MS/MS.Results1 The combination XCHT with CPT-11 can attenuate the toxicity of CPT-11 without affecting the antitumor acticity.1.1 XCHT and its main components have no significant effect on the antitumor activity of CPT-11.Relative tumor inhibition rate between CPT-11 group and CPT-11 combination with XCHT group or loperamide were not significantly different(the inhibition rates vary from 55.6 to 67.8%).1.2 XCHT and its components diminished the CPT-11 induced ITIXCHT and its main components significantly reduced the incidence and severy of irinotecan induced diarrhea.Co-administration of XCHT ameliorated the incidence of CID from 100%to 20%,without 3-4 degree diarrhea.Taken together,the ITI of CPT-11 group was sharply increased because of CPT-11 injection,and reached the maximum on day 8 and day 9.XCHT(3600 and 900 mg/kg)decreased the incidence for both fecal blood and severe diarrhea from day 6 to day 9(P<0.05,Wilcoxon rank sum test).Pretreatment with XCHT also markedly decreased the ITI of the mice compared with CPT-11 alone group(8.10±1.2 vs.4.1±0.99,P<0.05).Low does XCHT(225mg/kg)or loperamide,however,did not attenuate the toxicity of CPT-11.Baicalin and wogonin were showed strongly antidiarrheal activity,but the antidiarrhea activity of the wogonin combination with zingerone was best(ITI,3.2±0.19).Histopathological examnation showed that pretreat XCHT improved the damage of irinotecan to gastrointestinal tract.1.3 XCHT inhibits CPT-11-triggered inflammationCPT-11 alone increased the levels of TNF-?,IL-1?,IL-6 and IL-2 in the intestinal tissue and plasma as measured on 6 and 9 days of the treatment.XCHT signicantly reduced the expression of the TNF-?,IL-1?,IL-6 in intestine and plasma.1.4 XCHT combination with CPT-11 obviously decreased the concentrations of CPT-11,SN-38 in intestine and colon,but increased the concentrations of SN38-G in intestine.Compared with CPT-11 alone group,the SN-38 and CPT-11 concentrations in the ileum tissues from XCHT-pretreated mice were decreased to 71.7,88.3%(p<0.05)at 24h,respectively.No differences were found in the intestinal distributions of CPT-11 and SN38 between the two groups at 1h and 6h.However,XCHT significantly increased the concentrations of SN38-G at 1h and 6h to 2 and 1.65-fold.The colonic concentrations of CPT-11,SN-38 and SN38-G in CPT-11+XCHT group were lower than CPT-11 alone group.2 CPT-11 selectively impaired the expression of Ugts enzyme in the small intestine,the expression of Ugts were increased significantly with the co-administration XCHT.The protein expression of Ugt1a1 was decreased by 6 folds,Ugt1a6a and Ugt1a7c decreased by 10-folds,Ugt1a9 was lower than the quantitative lower limit,Ugt2a35 decreased by 2.3-folds in FVB mice.The XCHT combination with CPT-11 can significantly reduce the damage of the intestinal expression of Ugts,which was related to toxicity of CPT-11.The mechanism of XCHT may be related to induction of intestinal Ugts expression.However,there was no significant change in the expression of Ugts enzymes in the liver.CPT-11 also had no significant effect on the expression of Cyps in the liver and intestine.3 SN38 impaired the UGTs expression in Caco-2 cells,pretreat XCHT and wogonin:zingerone significantly induced the UGTs expression.The UGT1A1 levels were increased by 1.7~1.9-fold after treatment of Caco-2 cells with XCHT.The components of XCHT.Wogonin:zingerone significantly increased the UGT1A1 by 1.5~1.7 folds in a dose dependent manner.4 CPT-11 decreased the catalytic activity of intestinal Ugts of mice4.1 The concecutively administration of irinotecan significantly inhibited the activity of Ugt1a1 and Ugt1a9 in S9 miscrosome.The results showed that the activities of Ugt enzymes in intestinal S9 were decreased gradually after 6 days of consecutively injection of irinotecan.The activities of Ugt1a1 and Ugt1a9 were measured using estradiol and propofol as probe substrate.The activities of Ugt1a1 and Ugt1a9 reached bottom on day 4 and hadn' t returned to normal levels at day 9.The glucuronidification rates of SN38 were decreased from 200 pmol/mg/min to 1.04±0.08~2.36±0.07 pmol/mg/min,and decreased by 80-folds.However,the activity of Ugt1a1 and Ugt1a9 in the liver S9 was no significant difference during the experiment.The CPT-11 induced diarrhea occurred on day 6 and lasted to day 9.Therefore,it is indicated that the acticities of Ugts can be used as a marker to predict the toxicity of CPT-11.4.2 The XCHT had significantly changed the expression and activity of UGTs in the intestinal tract.Comparing with the CPT-11 alone group,the combination of XCHT with CPT-11 can significantly increase the glucuronidation velocity of SN38 from day 4 to day 6,and returned to normal levels on day 9.Although XCHT can't prevent the damage of SN38 to intestinal cells and UGTs enzymes,but XCHT could significantly induce the expression of UGTs,which reducing the toxicity by detoxicition of SN38.Correlation analysis between the expression and activity of UGT enzymes showed that the total protein level of intestinal S9 irrelevance of the expression of Ugts or Ugt isomers.The correlation between the expression and activity of Ugts was stronger than that between expression and activity of Ugt isomers(Ugts=Ugt1a1+Ugt1a6a+Ugt1a7c+Ugt1a9).It is suggested that SN38 dotoxication was involved in a variety of Ugt isoforms in mouse intestine.5 We provide a knockout ABC efflux transporters mouse models to help us to gain insight into the in vivo function of these transporters,especially BCRP.Bcrp dominate impact on the AUC of SN38 in the plasma which is related to the therapeutic efficacy,and Css of SN38 in intestinal exposure which is directly related to irinotecan-induced diarrhea.The correlation between the intestinal Css of SN38 and diarrhea was stronger than that between the AUC of SN38 and diarrhea.Multivariate analysis revealed that the Bcrp were significant variables for the intestinal and system exposure of SN38.The result was consistent with clinical practice which patients homozygous for the variant allele(ABCG2 421C>A)showed very extensive accumulation of SN38 and SN38-G and developed very severe side effects.However,pharmacokinetic parameters of CPT-11 and SN-38 were not significantly different between patients wild type for ABCG2 and patients carrying at least one defective ABCG2.The relation between the ABCG2 polymorphism and expression and function of BCRP also need to be elucidated.Therefore,the stratification of colon cancer patients,receiving irinotecan,based on these ABCG2 genotypes may be helpful for decreasing adverse effects and improving tumor responses6 CPT-11 also selectively impaired the intestinal expression of Ugts enzyme in Bcrp1(-/-)knockout mice.The protein expression of Ugt1a1,Ugt1a6a,Ugt1a7c,Ugt1a9 were decreased by 2~7-folds in Bcrp1(-/-)knockout mice.But there was no significant difference between FVB and Bcrp1(-/-)in Ugts expression after 6 days administration.There was also no significant change in the expression of Ugts enzymes in the liver of Bcrp1(-/-)knockout mice.CPT-11 also had no significant effect on the expression of Cyps in the liver and intestine.Both the BCRP deficient and CPT-11 caused Ugts enzyme damage contributed to the CID in Bcrp1(-/-)knockout mice,which resulted in more severe gastrointestinal toxicity in BCRP1(-/-)knockout mice than in wildtype FVB mice.ConclusionsThe XCHT combination with irinotecan can maintain the efficacy and reduce the gastrointestinal toxicity of CPT-11.Baicalin and wogonin were showed strongly antidiarrheal acttivities.Both efflux transporters and the UGTs related to the CID.Bcrp was predominantly contribution to the disposition and gastrointestinal toxicity of CPT-11.CPT-11 selectively impaired the expression and catalytic activity of intestinal Ugts enzyme in the mouse.XCHT combination with CPT-11 improved the SN38 glucuronidate activity and expression of Ugts in the liver and intestines of mice.XCHT reduced intestinal exposure of SN38,which is related to CPT-11 induced diarrhea,predominant due to accelerate detoxicification of SN38 to inactive SN38-G by intestinal Ugt enzymes.The interplay of effluxs transport deficient and impaired ugts enzymes cuased by CPT-11 in the small intestine can aggravate CID.This study provides a theoretical and practical basis for XCHT combination with CPT-11 in clinical practice. |
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