Drug Concentrations And Pharmacodynamic Analysis Of Osimertinib And Recombinant Human-murine Chimeric Anti-CD20 Monoclonal Antibody In Cancer Patients

In this study,we aimed to assess the concentration and safety of osimertinib and recombinant human-murine chimeric anti-CD20 monoclonal antibody(SCT400)in cancer patients.A method for the determination of Osimertinib in human plasma and cerebrospinal fluid(CSF)using ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was developed and fully validated.The gene variation of circulating tumour DNA(ctDNA)in plasma and cerebrospinal fluid were detected by next-generation sequencing(NGS).An enzyme-linked immunosorbent assay(ELISA)was established and validated for detecting the concentration and immunogenicity of SCT400 and Rituximab in human serum.Meanwhile,UPLC-MS/MS method was also developed for detecting concentration of SCT400.Results of present study were utilized to analyze pharmacokinetic(PK)and pharmacodynamic(PD)for above drugs.The main content was divided into two parts:Section 1.Osimertinib quantitative and gene variation analyses in plasma and CSF of non-small cell lung cancer patient with leptomeningeal metastasesPatients with Leptomeningeal metastases(LM)have rapid progression,poor treatment effect,and short survival time.Osimertinib,a third-generation EGFR-TKI has promising efficacy for LM.Chapter 1:Development and validation of the UPLC-MS/MS method for determination of Osimertinib in human plasma and cerebrospinal fluid from NSCLC with LM.Protein precipitation was used for sample preparation.The calibration range of UPLC-MS/MS method was 2-500 ng/mL in plasma and 0.5-20 ng/mL in cerebrospinal fluid.The intra-and inter-day precision values were 1.96%-9.34%and 3.04%-9.73%,respectively,and the accuracy values for detecting osimertinib were-13.40%-4.40%in plasma and-9.75%-3.13%for CSF at four concentration levels.The mean relative recoveries of the QC samples were 101.5%and 103.5%in the plasma samples,and 105.7%and 94.9%in the CSF samples.No significant matrix effect was observed.Osimertinib was stable under normal laboratory conditions.The method was successfully applied to detect osimertinib in dynamic 4 plasma samples and 8 CSF samples from a NSCLC patient with LM after EGFR-TKI failure.The concentrations of osimertinib in plasma and CSF were 105.13 ng/mL and 1.60 ng/mL,respectively.The deviation ratio for the CSF-plasma concentration(penetration rate)was 1.47 ± 0.3%.Chapter 2:Use next-generation sequencing to detect the gene variation of ct DNA in plasma and CSF from patient with LM NSCLC.ctDNA derived from plasma and CSF was performed with a panel of 1021 genes.Mutations were detected in mTOR,EGFR,CHECK1,ABCC11,and TP53 of ctDNA from plasma and CSF samples.The average variations allele frequency(VAF)of mTOR was the highest among these.The detected mutation rate of CSF samples was higher than that of plasma samples(50%vs25%).Quantitative analysis of molecular tumor burden in cerebrospinal fluid samples using mTBI was(15.62%,3.93%,13.16%and 16.41%).Our data further revealed that VAF and mTBI of ctDNA derived from CSF exhibited negative correlation with treatment effect,the correlation of osimertinib concentrations and treatment effect in CSF was positive.ctDNA derived from CSF may be served as a useful biomarker for monitoring the treatment effect for LM.Section 2.Analysis of drug concentration and immunogenicity of recombinant human-murine chimeric anti-CD20 monoclonal antibody(SCT400)and rituximab injection in human serumSCT400 is a chimeric anti-CD20 antibody made in China that is similar to Rituximab.It was constructed by IgGl? constant regions and murine anti-CD20 monoclonal antibody variable region.SCT400 presents the consistent pharmacology and toxicology profile with Rituximab in preclinical and phase I clinical trail.To further evaluate the similarity,the comparison of the pharmacokinetic properties in CD20 positive non-Hodgkin's lymphoma patients with the same dosage.Chapter 1:Development and validation of two analytical methods for determination of anti-CD20 monoclonal antibody in human serum.The calibration range of ELISA method was 1.56-50 ng/mL in serum.The intra-and inter-day precision values were 3.52%-16.32%and 1.74%-22.55%,respectively,and the accuracy values for detecting osimertinib were-19.70%-8.14%and-22.02%-15.50%for SCT400 and rituximab at five concentration levels.No significant matrix effect was observed.SCT400 and Rituximab were stable under different laboratory conditions.The method has good parallelism.The method was successfully applied to perform serum pharmacokinetic studies of SCT400 and rituximab in phase II trail.With the extensive using of UPLC-MS/MS in detection of drug concentrations,we further explored its superiority in detection of monoclonal antibody drugs.We used the enzymolysis technology to screen out the characteristic peptides of SCT400.A method for the determination of SCT400 and Rituximab concentration in human serum by UPLC-MS/MS was preliminarily established.Part of validation was completed and the consistency of the two methods was evaluated.Chapter 2:Development and validation of sensitive ELISA method for immunogenicity of SCT400 and rituximab in human serum.The calibration range of ELISA method was 3.13-100 ng/mL in serum.The intra-and inter-day precision values were 0.73%-15.47%,and the accuracy values were-9.53%-13.02%for positive antibody at three concentration levels of coating SCT400;The intra-and inter-day precision values were 1.54%-16.84%,and the accuracy values were-9.13%-12.36%for positive antibody at three concentration levels of coating Rituximab.No significant matrix effect was observed.Positive antibody was stable under different laboratory conditions.The results of drug resistance showed that in the presence of positive antibody at a concentration of 80ng/mL,both SCT400 and rituximab were able to tolerate a concentration of 12.5?g/mL with a Mol.Ratio of 1:156.At a concentration of 8 ng/mL,SCT400 and rituximab were able to tolerate a concentration of 3.125 ?g/mL and 1.5625 ?g/mL with Mol.Ratio of approximately 1:390 and 1:195.The method was successfully applied to perform serum immunogenicity of SCT400 and Rituximab in phase II trail.The clinical samples were screened for positive antibodies and further confirmatory tests were performed to confirm the results,and antibody titer was detected.