| Dexlansoprazole belongs to the proton pump inhibitor,is a new drug developed by Japanese Takeda pharmaceutical for the treatment of esophageal inflammation.The drug could prolong the time of drug action,increase patient compliance and has obvious clinical curative effect through the double delayed release(DDR)system.It became the focus of imitation due to the patent protection expires in 2020.QO-58 is a new KCNQ type potassium channel opener obtained throughput screening.The preliminary study of pharmacodynamics indicated that it has the effects of anti convulsion,anti epilepsy and analgesia,its lysine(QO-58 lysine)improves absorption and boosts efficacy.On the basis of previous pharmacological,toxicological,and part of the toxicokinetics,the study of metabolism and excretion in vivo provides experimental basis for the research and development of new drugs.LC-MS/MS combined the separation of liquid chromatograph and the detection of mass spectrometric with strong separation ability,high sensitivity and specificity,has become an important means of drug discovery as well as in vitro and in vivo drug analysis.In this study,the LC-MS/MS technology was used to assess the domestic high imitation and Dexilant products(Takeda dexlansoprazole,sustained-release capsules)in bioequivalence of Beagle dogs.At the same time,the metabolism and excretion of QO-58 lysine in rats were also studied.The results provide an important experimental basis for further development of QO-58 lysine.Part one The comparison of two kinds of Dexlansoprazole sustained-release capsules' bioavailability in Beagle dogsObjective: To study if the test drug and the reference drug of dexlansoprazole have bioequivalence in beagle dogs.Methods:1 Sample collection: The randomized crossover design was used in this experiment,feeding 30 mg dexlansoprazole sustained-release capsules in each cycle.Blood samples were collected from the leg veins of the Beagle dogs to vacuum heparin tube,and samples were collected before and after administration of 0.5,1,1.5,2,2.5,3,4,5,6,8,10,12 h.2 Sample pretreatment: Liquid liquid extraction(ethyl acetate)was used for sample pretreatment.3 Test conditions: Phenomenex C18 column was used,and the mobile phase was methanol(A): 2 m M ammonium acetate solution(B),using gradient elution.The MRM transitions from precursor ions to product ions were optimized as m/z 369.8?252.0(Dexlansoprazole)and 345.9? 198.0(omeprazole,internal standard).4 Bioequivalence: After the pharmacokinetic parameters were calculated,the AUC0-t,AUC0-? and Cmax were converted to logarithm,and the two one-sided t-test was used to determine whether the two formulations were bioequivalent.Results:1 The method was validated for good linear relationship(r?0.9960),inter-day,intra-day precision and stability could meet the requirements,and have no significant matrix effect.2 Single dose of test drug and reference drugs were given,the main pharmacokinetic parameters(AUC0-t,AUC0-? and Cmax)after logarithmic transformation and carry on the two one-sided t-test.The results showed that there was no significant difference between the two preparations.Conclusions:The LC-MS/MS method established in this study is simple and reliable,which could meet the requirements of biological sample analysis.Two kinds of Dexlansoprazole sustained-release capsule have similar process in vivo and the pharmacokinetics in Beagle dogs,the two preparations are bioequivalent.Part two Excretion kinetics and the determination of QO-58 lysine in rats' urine,feces and bile by liquid chromatography coupled with mass spectrometryObjective: To establish a liquid chromatography coupled with mass spectrometry(LC-MS/MS)method for the determination of QO-58 lysine in urine,feces and bile of rats,and to study its excretion kinetics as well.Methods:1 Sample collection: QO-58 lysine was intragastric administrated in the dose of 50 mg/kg,and the samples of urine and feces were collected by metabolic cages at 0-4,4-8,8-12,12-24,24-36,36-48,48-60 and 60-72 h.Rats were treated with bile duct intubation under anaesthesia.After suture,collect bile samples at 0-4,4-8,8-12,12-16,16-20,20-24,24-28,28-32,32-36,36-40,40-44,44-48,48-52,52-56,56-60,60-64,64-68,68-72 h in sober state.2 Sample pretreatment: Liquid-liquid extraction method was adopted for urine and bile.However,it is suitable to use acetonitrile precipitation method for feces.3 Test conditions: Phenomenex C18 column was used.Acetonitrile(A)and 2 mmol/L of ammonium acetate and 0.2%(v/v)formic acid solution(B)was taken as mobile phase and flow rate was 0.5 ml/min.The electrospray ionization ion source,positive ion mode and multiple reaction monitoring were used to detection,and the internal standard substance(IS)was Nimodipine.The MRM transitions from precursor ions to product ions were optimized as m/z 443.1?361.1 for QO-58 lysine,m/z 419.2?343.2 for Nimodipine(IS).Results:1 The method was validated for good linear relationship(r?0.9943),inter-day,intra-day precision and stability could meet the requirements,matrix effect between 69.48%-99.57%.2 The cumulative excretion rate of 72 h was(0.0245 ± 0.0324)% for urine,(92.94 ± 4.71)% for feces and(2.05 ± 0.68)% for bile,respectively.Conclusions:1 In this experiment,LC-MS/MS method was taken to determinate the content of QO-58 lysine in urine,feces and bile.The method is simple,rapid,and has good specificity,stability and accuracy.The methodological study exhibited that could meet the demands of sample determination.2 From the result we could observe that QO-58 lysine after intragastric administration was mainly through feces excretion,part via bile excretion and few from urine excretion.In addition,about 7% of QO-58 lysine remains to be proven,it could produce some new metabolites in liver or other organs.Part three Metabolism of QO-58 lysine in ratsObjective: To establish LC-MS/MS and HPLC-QTOF-MS methods,separation,screening,speculation and analyse QO-58 lysine metabolites in rat urine and bile.Methods:1 Phenomenex C18 analytical column was used.Acetonitrile(A)and 2 mmol/L of ammonium acetate and 0.2%(v/v)formic acid solution(B)was taken as mobile phase and gradient elution was also used.Mass spectrometry used electrospray ion source.2 Rats were given QO-58 lysine with 50 mg/kg through intragastric administration.Urine and bile samples were collected,concentrated and treated by liquid-liquid extraction.High performance liquid chromatography quadruple time-of-flight mass spectrometry was taken to analyze samples.Positive / negative ion mode,full scan acquisition were also used.3 After data collection,use “Fragment and Neutral defect filter” function in “Metabolitepilot 1.5” to carry on the preliminary screening for these metabolites.Then use “Masterview” function in “Peakview 2.2”,further analysis was performed on the results of the preliminary screening.The exact molecular weight,molecular formula,MS2,retention time and mass spectrum of the compounds were compared and speculate on their structure.Finally,the metabolic pathway was conjectured according to the metabolite structure.Results:Eleven metabolites were detected in the rat according to the law of metabolic transformation of drugs in vivo and the lysis of the parent drug.Among them,8 kinds of phase I metabolites and 3 kinds of phase II metabolites were found.And the metabolic pathways of each metabolite were analyzed.Conclusions:1 A total number of 11 metabolites were detected in this study.QO-58 lysine's phase I metabolites could occur the reaction of halogen atoms elimination,hydroxylation and dihydroxylation.The phase II metabolites can undergo sulfation,glucuronidation and N-acetylcysteine conjugation.2 Established a metabolite identification method based on HPLC-QTOF-MS technique which has good specificity,high accuracy,convenient and quick. |
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