Metabolic Characterization And Pharmacokinetics Of Sutetinib In Cancer Patients

Backgroundmetabolites are often closely related to the safety and effectiveness of drugs.Therefore,in the clinical development stage of innovative drugs,we hope to obtain human metabolite data as soon as possible,especially the metabolite spectrum in plasma,so as to determine the main metabolites in the systemic circulation,and carry out pharmacokinetic evaluation together with the parent drugs.Sutetinib,a new type of covalent tyrosine kinase inhibitor,has completed the clinical phase I study.Objective Identify the main metabolites of sutetinib in the plasma of tumor patients,and establish of a sensitive and accurate liquid chromatography-tandem mass spectrometry method for simultaneous determination of sutetinib and the main metabolite in human plasma.Explore the stability conditions of parent drug and major metabolite in whole blood and plasma and provide a reference for clinical biological sample collection.Apply the validated method to the pharmacokinetic study of sutetinib in tumor patients.Method ultra performance liquid chromatography-UV/triple time-of-flight mass spectrometry?UPLC-UV/Q-TOF-MS?technology was used to identify metabolites in the plasma of tumor patients after a single oral administration of 60mg Sutetinib capsules.A LC-MS/MS method was established for the simultaneous determination of sutetinib and its main metabolites,with stable isotope-labeled d6-sutentinib and d6-N-oxide sutentinib as internal standards.The analytes and internal standards were extracted from plasma by protein precipitation with acetonitrile,and separated on a Agilent XDB C₁₈?4.6×50 mm,1.8?m?column using a isocratic elution procedure.The mobile phase consisted of acetonitrile and 2mmol·L^-11 ammonium acetate?0.01%ammonia??57:43?at a flow rate of 0.5mL·min^-1.Positive electrospray ionization was performed using multiple reaction monitoring?MRM?with transitions of m/z 440.2?367.1 for sutetinib and m/z446.2?367.1 for its internal standard d₆-sutetinib,m/z 456.3?396.2 for N-oxide sutetinib and m/z 462.3?396.2 for its internal standard d₆-N-oxide sutetinib.Result After a single oral administration of 60 mg sutetinib,a total of 11metabolites were detected in the plasma in addition to the parent drug.The main metabolite was N-oxidation?M6-3?.Other metabolites included N-dealkylation?M1?,N-bisdemethylation with formylation?M5?,mono-oxidation?M6-1 and M6-4?,Alkynyl oxidation to carboxylic acid?M7?,oxidative deamination to carboxylic acids?M13?,double oxidation?M14?,alkynyl oxidation to carboxylic acid with mono-oxidation?M16?and glutathione binding?M22-1 and M22-2?.Both sutetinib and N-oxide sutetinib quantitative analysis showed good linearity in the range of 0.50¹00 ng·mL^-1.Correlation coefficient?r?was greater than 0.99.The intra-day precision of sutetinib was less than 3.9%,the intra-day precision was less than 5.4%,and the accuracy was between 3.8%and 4.7%;the intra-day precision of N-oxide sutetinib was less than 4.0%,and the intra-day precision was less than 6.2%,The accuracy was between 2.8%³.8%.The results of the plasma stability study revealed that the concentration of sutetinib in plasma decreased by approximately 29%and 66%after being placed at room temperature for 6 and 19 hours,respectively.UPLC/Q-TOF MS detection proved that it was covalently bound to human serum albumin?HSA?in plasma,and a higher degree of protein binding with increasing temperature.The N-oxidation in plasma was stable at room temperature for 6 and 19 hours.The N-oxidation with plasma was incubated at room temperature for 2,8,and 24 hours and at 37°C for 2,8 hours there had no covalent conjugate was detected,while at 37°C for 24 hours there had conjugate.Compareing with the parent drug,the covalent binding with HSA of N-oxidation is significantly reduced,which may be due to the introduction of O atoms,the electrophilicity of the compound changes,and the affinity with the HSA binding pocket decreases.At the same time,the introduction of O atom changes the molecular structure and increases the steric hindrance.Therefore,the whole blood sample collection process should be kept at the wet ice and does not exceed 2 h,and the plasma sample pretreatment should be kept at wet ice for no more than 5 h or room temperature for no more than 2 h.Cancer patients after oral administration of 40-100 mg sutetinib,the absorption was rapid,the average peak time is 2.17 h,and the average peak time of N-oxidation was slightly delayed by 3 h.Within the dose range of 40-80 mg,AUC_0-t-t and C_maxax of sutetinib increased with the increase of the administered dose,which was basically proportional to the dose.The plasma exposure of the metabolite is 1.01¹.44 times of the parent drug.The average plasma elimination half-life of sutetinib is 16.6 h,and the average plasma elimination half-life of major metabolite N-oxidation sutetinib is 16.0 h.Conclusion The metabolites of sutetinib in the plasma of tumor patients were identified for the first time.A chromatography-tandem mass spectrometry?LC-MS/MS?method was developed and validated for the simultaneous determination of sutetinib and its N-oxide metabolite in human plasma.The method was successfully applied to the pharmacokinetic study of sueitinib in tumor patients.The plasma exposure of N-oxidation was significantly higher than that in the pre-clinical safety assessment species,suggesting that the clinic should pay attention to the effect of this metabolite on drug efficacy and safety.The results of stability study indicated that clinical sample collection and pretreatment should be carried out at low temperature.