| Objective:The quassiniod brusatol,is one of the main active ingredient that isolated from the traditional Chinese medicine Bruceajavanica.Brusatol has a wide variety of anticancer,antiprotozoal,anti-inflammatory,antiphytovirus and antifedant pharmacological activities.The anticancer effect was most studied to identify the mechanisms,which include inhibition of protein synthesis,activation of NF-κB pathway,down-regulation of C-myc protein level,and inhibition of Nrf2 pathway by promoting ubiquitination.The latest study acknowledged brusatol as a unique inhibitor of Nrf2 pathway,show the activity of reduce tumor burden and ameliorate chemoresistance in both in vitro and in vivo models.This indicates that brusatol has great potential to develop into novel chemotherapy drug,and makes brusatol a hotspot.Detailed knowledge about thhe pharmacokinetics of brusatol(i.e.5 absorption,distribution,metabolism and excretion)is essential for further drug development,although such research is still scarce.Therefore,it is imperative to systematicallystudy and understand the pharmacokinetics of brusatol.This project focus on the in vivo process of brusatol,systematically studied the pharmacokinetics,tissue distribution,metabolites and excretion process.At the same time,the effects of brusatol on the plasma pharmockinetic,tissue distribution and nephrotoxicity of cisplatin were investigated.All this study can promte the development progress of brusatol as an chemotherapy sensitizer drug.Methods:1.Pharmacokinetics and tissue distribution of brusatol after single dose administration in miceA sensitive HPLC-MS method was established and validated for the quantification of brusatol in plasma and tissues.The method was validated in compliance with the NMPA guidelines for bioanalytical method validation,including selectivity,matrix effect,linearity,recovery,accuracy and precision,dilution integrity,and stability evaluations.Animals were randomly divided into three dosage groups,each group received brusatol at a dose of 1 mg/kg,1.5 mg/kg and 2 mg/kg respectively.Blood was collected through the eyeball at 0,3,5,10,15,30,60,90,180 and 240 min after administration.The animals was then sacrificed and dissected to collect the liver,kidney,lung,and spleen.The validated method was successfully applied to determine the concentration of brusatol in plasma and tissues,pharmacokinetic parameters of brusatol in each group were calculated by using DAS2.0 respectively.2.Metabolism and excretion of brusatol in rats after single dose administrationA sensitive HPLC-MS/MS method was established and validated for the quantification of brusatol in plasma,bile and urine.The LC-MS/MS method was validated according to the National Medical Products Administration(NMPA)guidelines for bioanalytical method validation.24 rats were randomly divided into three groups:plasma group,bile group and urine and feces group.In each group,six rats were administered brusatol solution via the tail ven at a single dose of 1 mg/kg,while the other two were administered the corresponding volume of blank solution(0.9%saline with 1%DMSO)only.Blood was collected at predose and 5,10,15 and 30 min,and 1,2,3,6 and 8 h post dose.The rats in bile group were anesthetized with 3%pentobarbital sodium,then an abdominal incision was made,and the common bile duct was cannulated with polyethylene tubing to collect the bile samples.After 20 min of stable outflow of bile,rats were administered brusatol or an equal volume of blank solution.The bile sample were collected during 0-1,1-2,2-3,3-4,4-5,5-6,6-7,7-8,8-9,9-10,10-11 and 11-12 h after the administration.The rats in urine and feces group were kept separate in stainless steel metabolic cages.Blank urine and feces were collected before drug administration and urine samples were collected 0-2,2-4,4-6,6-8,8-10,10-12 and 12-24 h after administration.The validated LC-MS/MS method was successfully applied to the pharmacokinetic and excretion study;the high resolution mass spectrometry using a Thermo Q Exactive Focus was applied for the identification of metabolites.The raw data files obtained from the Thermo Q Exactive Focus were imported into the Compound Discoverer(CD)software to identify metabolites of brusatol.3.Effect of brusatol on pharmacokinetics and tissue distribution of cisplatinA sensitive HPLC-MS/MS method was established and validated for the quantification of cisplatin in plasma and tissues.The method was validated in compliance with the NMPA guidelines for bioanalytical method validation,including selectivity,matrix effect,linearity,recovery,accuracy and precision,dilution integrity,and stability.The mice were randomly divided into the cisplatin group,and the combination group.The mice in cisplatin group were intraperitoneally injected with cisplatin only(10 mg/kg),and the mice in combination group were injected with 2 mg/kg of brusatol through the tail vein half an hour before the administration of cisplatin.Blood and tissues samples were collected at 5 min,10 min,15 min,30 min,1 h,2 h,4 h,8 h,12 h,24 h after cisplatin injection.The validated method was used to determine the concentration of cisplatin in plasma and tissues,pharmacokinetic parameters of cisplatin in each group were calculated by using DAS2.0 respectively to compare the difference with or without brusatol.4.Effect of brusatol on the cisplatin-induced nephrotoxicityKunming mice were intraperitoneally injected with cisplatin(10 mg/kg)to induce nephrotoxicity.The mice in combination group were pretreated with brusatol(1mg/kg or 2 mg/kg.)for three days,then cisplatin(10 mg/kg)was injected to induce nephrotoxicity.Two days later after last administration,the blood samples were collected to monitor the level of BUN and CR.The kidneys were collected to determine the activities of GSH、SOD、CAT、MDA、NO and NOS;HE were uesd to observe the histopathological changes.Western blotting was used to detected the expression of Nrf2、iNOS、caspase-3、TN F-a、IL-6,in order to explore the underlying mechanism.Results:1.Pharmacokinetics and tissue distribution of brusatol after single dose administration in miceA highly sensitive,accurate and reproducible LC-MS method for simultaneous quantification of brusatol in plasma and tissues was developed and validated.Pharmacokinetic and distribution studies were carried out by using this method.The plasma concentration of brusatol decreased rapidly with the half-life time in 10 min,the concentration in tissues increased rapidly and reached the peak in 15-30 min after the tail vein injection,then slowly eliminated and can still be detected until 4 h.It should be noted that the peak concentration of brusatol in lung was 10 fold or more higher than that in other tissues,suggested that brusatol may actively target at lung.2.Metabolism and excretion of brusatol in rats after single dose administrationThe plasma pharmacokinetic and elimination of brusatol in rats after intravenous dosing was studied using an efficient and sensitive HPLC-MS/MS method.With the half life time(tl/2)<30 min,the plasma concentration decreased rapidly.The average cumulative excretion rate of brusatol was 5.82%in urine during 24 h,and 0.71%in bile during 12 h.With the help ot Thermo Q Exactive TM Focus and Compound Discoverer,four metabolites were detected and identified.M438 is a hydrolysis product of brusatol,which can be detected in bile,urine and feces;M536 is a hydroxylation product of brusatol,and the abundance is higher than that of other metabolites and even brusatol in all of the matrices.Therefore,hydroxylation is supposed to be the major metabolic pathway of brusatol in rat.M618 and M424 are only detectable in urine,M618 is a glucuronidation product and M424 is the demethylation or hydrolysis product of M438.owing to the lack of an MS2 spectrum of M424,the structure cannot be finalized.3.Effect of brusatol on pharmacokinetics and tissue distribution of cisplatinThe HPLC-MS/MS established in this section can be used for the simultaneous determination of cisplatin in plasma and tissue homogenates.With the liner range of 5-3000ng/ml in plasma and 5-1200ng/ml in tissues,this method shows good precision and accuracy,can meet the requirements for biological sample testing.The peak concentration of cisplatin(10mg/kg)after intraperitoneal administration was 5326.68 mg/L,with the combination of brusatol,the peak concentration was decreased to 4784.05 mg/L,AUC0-t was decreased significantly at the same time,but the volume of distribution was increased.It suggests that more drugs distributed into the tissues.4.Effect of brusatol on the cisplatin-induced nephrotoxicityThe body weight of the mice in the cisplatin group and the combination group showed a downward trend,futher weight loss was observed in the combination group than that in the cisplatin group.In cisplatin group,with the increase of BUN and CR,a significant deterioration of renal function was observed two days after the intraperitoneal injection.While the BUN and CR levels were further increased with the pretreatment of brusatol.HE staining showed that the combination group had more tubular epithelial cell necrosis and cortical medullaiy congestion compared with cisplatin group.The use of cisplatin caused a decrease in the level of GSH,SOD,CAT,and an increase in MDAlevel;there are also an increase of the activity of NOS and the level of NO.The GSH value of the combination group was lower than that of the blank group,and increased when compared with the cisplatin group,but the increase was not statistically significant.The activity of SOD and CAT enzymes futher decreased compared with the cisplatin group,and the MDA level was futher increased at the same time.Combination with brusatol led to more increase of NOS,as a result,the NO level was futher increased.The expression of Nrf2 protein in the brusatol group and the combination group was significantly lower than that in the blank control group and the cisplatin group alone.The protein expression of iNOS in the cisplatin group was significantly higher than that in the blank control group and the brusatol group,it was further increased in the combination group.A marked accumulation of pro-inflammatory cytokines TNF-a was observed in the siaplatin group,with the combination of brusatol,the expression of TNF-a showed a downward trend,but it was still significantly increased compared with the blank group and the BRU group.Conclusions:1.This is the first reported LC-MS method for detecting brusatol in tissues and can accurately determine the concentrations of these compounds in plasma and different tissues.The concentration of brusatol in plasma decreased rapidly but slowly eliminated and can still be detected until 4 h in tissues.A more than 10 fold concentration of brusatol was found as compared to that in other tissues.2.The average cumulative excretion rate of brusatol were both low in urine and bile,and four metabolites were identified and rationalized,hydroxylation is supposed to be the major metabolic pathway of brusatol in rat.3.The combination with brusatol reduced the plasma concentration of cisplatin and increases the volume of distribution;at the same time,the concentration of cisplatim in kidney and lung were increased,especially in kidney,the increase was significantly.4.Compared with the cisplatin group,the BUN and CR in serum were increased in the combination group,also the anti-oxidation index in tissue were decrease and the inflammatory factors were increased;pathological findings showed increased tubular congestion.All this results suggested that brusatol caused the cisplatin-induced nephrotoxicity further aggravated. |
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