Metabolic Characterization And Pharmacokinetics Of Bisthianostat In Cancer Patients

Background Metabolites are often closely related to the safety and efficacy of drugs,so the data of metabolites,especially in human plasma,should be obtained as soon as possible in the clinical development stage of innovative drugs,so as to determine the main metabolites in systemic circulation,and conduct pharmacokinetic study together with prototype drugs.Bisthianostat is a novel histone deacetylase（HDAC）inhibitor with a new structure.It is an anti-tumor drug in clinical trials developed by Shanghai Institute of Materia Medica,Chinese Academy of Sciences.Objective The main metabolites of bisthianostat in human plasma were identified,and a bioanalytical method for simultaneous determination of bisthianostat and its main metabolites in human plasma by liquid chromatography-tandem mass spectrometry（LC-MS/MS）was established.The method was validated and applied to the pharmacokinetic evaluation of bisthianostat and its metabolites,providing a basis for the development of bisthianostat.Method Metabolites were identified by ultra performance liquid chromatography-UV/triple time-of-flight mass spectrometry（UPLC-UV/Q-TOF-MS）in the plasma of cancer patients after a single oral administration of 100 mg bisthianostat tablet.Using stable isotope-labeled of d₄-bisthianostat and d₅-M351 respectively,after extraction from the plasma by protein precipitation in acetonitrile,the analytes were separated from endogenous substances on a column of Waters BEH C₁₈（2.1 mm×50 mm,1.7μm）.5 mmol·L^-1ammonium acetate（containing 0.2%formic acid,v/v）（pump A）and acetonitrile（pump B）for gradient elution to achieve the chromatographic baseline separation between bisthianostat and M351.Multiple reaction monitoring（MRM）was performed with transitions of m/z 367.1→235.0 for bisthianostat,m/z 352.1→207.0 for M351,m/z 371.1→235.0 for d₄-bisthianostat,and m/z 357.1→208.0 for d₅-M351 using positive electrospray ionization.The method was linear over the concentration ranges of 2.00-2000 ng·m L^-1for bisthianostat and4.00-4000 ng·m L^-1 for M351.The society of ethics agreed the hospital to conduct the clinical trial in Renji Hospital,School of Medicine of Shanghai Jiaotong University.Result After a single oral administration of 100 mg bisthianostat,a total of 14 metabolites were detected in the plasma in addition to the parent drug.The main metabolic pathways are N-hydroxyamide hydrolysis,other metabolic pathways include N-dealkylation（M1）,amide hydrolysis and oxidation（M2 and M3）,hydroxylamine reduction（M5）,N-acetylcysteine binding（M12-1 and M12-2）and glucuronic acid binding（M14）.In this paper,the LC-MS/MS method was established for the first time to determine the hydrolyzed metabolites of bisthianostat and its n-hydroxyamide in human plasma.The linear range of bisthianostat was2.00～2000 ng·m L^-1,and the linear range of N-hydroxyamide hydrolyzed metabolites was 4.00～4000 ng·m L^-1.The intra-and iner-day precision and accuracy of bisthianostat and its N-hydroxyamide hydrolytic metabolites in low,medium and high quality control samples all met the relevant requirements of biological sample testing.Conclusion It is the first time to identify the metabolites of bisthianostat in the palsma of cancer patients,and the results showed that N-hydroxyamide hydrolyzed metabolites are metabolites with a high proportion in human body,and their safety needs further attention.A rapid,sensitive and accurate LC-MS/MS method was developed for simultaneous determination of bisthianostat and N-hydroxyamide hydrolyzed metabolites in human plasma,which was verified by a complete methodology and successfully applied to the pharmacokinetic study of bisthianostat in the first clinical trial.Pharmacokinetic data show that After oral administration of 100 mg and 200 mg bisthianostat tablets,the patients were absorbed rapidly,and the peak time of the concentration of bisthianostat was 0.75 h,which was eliminated quickly in the body,and the half-life of plasma elimination was about 4.3 h.When the dose increased from 100 mg to 200 mg,the plasma peak（tmax）concentration and AUC were basically consistent with the ratio of increased dose.When the dose was increased to 400 mg,the growth rate of the C_max is not obvious.The average peak time of N-hydroxyamide hydrolyzed metabolite M351 in bisthianostat plasma was 3.7 h later than that of the parent.The half-life of plasma elimination was about 8.0 h.The plasma exposure of M351 in vivo was higher than that of the prototypical drug,and the AUC ratio was 11.6-15.2,suggesting that the influence of metabolites on the efficacy and safety of the drug should be paid attention to in the clinical study of bisthianostat.