Study On The Pharmacokinetics,Brain Tissue Distribution And Related Substance Methodology Of HBW-3-10 In Rats

Research background and purpose:Bruton’s tyrosine kinase（BTK）is an important signaling molecule in the B cell receptor pathway.It is involved in regulating the proliferation,differentiation and apoptosis of B cells,and plays an important role in the survival and proliferation of malignant B cells.BTK inhibitor is an important target for clinical research in the treatment of B cell tumors and B cell immune diseases.It is suitable for the treatment of chronic myelogenous leukemia（CLL）,acute myeloid leukemia（AML）,adult mantle cell lymphoma（MCL）and WM,and other B-cell malignancies.At present,the five BTK inhibitors on the market are all irreversible inhibitors,and patients with B-cell malignant tumors may develop drug resistance after 3 to 5 years of use,thus no longer playing a therapeutic role.There is a substantial clinical unmet need for new therapies to overcome the resistance and toxicity of irreversible BTK inhibitors.HBW-3-10 is a second-generation reversible BTK inhibitor developed and synthesized in our laboratory.It has good activity against the BTK C481S mutant kinase resistant to the first-generation irreversible BTK inhibitor without obvious off-target toxicity,and is a candidate compound for the treatment of B-cell malignant tumors.In order to further explore the medicinal properties of HBW-3-10,the pharmacokinetics,brain tissue distribution and related substance methodology of HBW-3-10 in rats were preliminarily studied.Materials and Methods:1.The LC-MS/MS method was established and validated to determine the concentration of HBW-3-10 in rat plasma.Plasma samples were treated by protein precipitation method.The determination was performed on Agilent Zorbax Eclipse C18（2.1 mm×50 mm,1.8μm）column with mobile phase consisted of 2mM ammonium formate-acetonitrile at the flow rate of 0.3 mL/min.The column temperature was 30℃.Using MRM mode for quantitative analysis,the quantitative ions were m/z 539.2→377（HBW-3-10）and m/z 465.2→183（HBW-3151,internal standard）,respectively.Rats were given intragastrically（ig）and intravenously（iv）,and blood samples were collected and measured at 5 min（iv only）,15 min,30 min,1 h,2 h,4 h,8 h,10 h and 24 h after administration,respectively.DAS 3.0 software was used to calculate pharmacokinetic parameters and draw drug-time curve.2.Develop and validate the LC-MS/MS method for HBW-3-10 analysis in rat biological samples（brain tissue and cerebrospinal fluid（CSF））.After intragastric administration,biological samples were collected at 0.25 h,1 h,2 h and 4 h after administration,respectively,and drug concentrations of HBW-3-10 in the biological samples and plasma were detected.3.HPLC analysis method for related substances of HBW-3-10 was established.A60min gradient elution method was established using ACE Excel 3 Super C18（4.6mm×150 mm,3μm）column as stationary phase and 50 mM sodium perchlorate-acetonitrile as mobile phase at the flow rate of 1mL/min and column temperature of 40℃.The impurity content was calculated by the peak area normalization method and the method was pre-validated.Result:1.The LC-MS/MS method for the determination of HBW-3-10 plasma concentration in rats was successfully established and verified.The specificity,residue,linearity and lower limit of quantification（LLOQ）,accuracy and precision,dilution reliability,matrix effect and extraction recovery,stability and other items of the method meet the requirements of validation of quantitative analysis methods in vivo.After ig administration,HBW-3-10 reached the maximum plasma drug concentration C_max（4344.207±1979.695）ng/mL,AUC_0-twas（17905.914±9798.088）ng/mL·h in rats at（0.5±0.274）h,elimination half-life t_1/2was（2.511±1.272）h.The oral bioavailability of HBW-3-10 was 13.37%±7.303%,and there was no significant difference in oral bioavailability between male and female rats.2.The LC-MS/MS analysis method for HBW-3-10 concentration in rat brain tissue and CSF was successfully established and verified.The specificity,residue,linearity and LLOQ,accuracy and precision,dilution reliability,matrix effect and extraction recovery,stability and other items of the method meet the verification requirements of quantitative analysis methods for biological samples.The maximum concentration of drug detected in brain tissue and CSF was（86.339±4.516）ng/g and（51.932±51.932）ng/mL,respectively.HBW-3-10 could be distributed in brain tissue through the blood-brain barrier（BBB）and CSF barrier.3.HBW-3-10 was slightly soluble in acetonitrile and in methanol,and had good stability within 24h in the sample solution under the condition of cold storage（2～8℃）.The blank solution does not interfere with the main peak,the known impurities and the exit peak of the degraded impurities.The known impurities do not interfere with the detection of the main peak.The separation between impurities is good,and the method has good specificity.Taking 0.10%of the test product concentration as the limit concentration of the impurity,the limit of quantification S/N was about 27.0,which was equivalent to 34%of the limit concentration of the impurity.The detection limit of S/N is about 6.1,equivalent to 8.5%of the impurity limit concentration.At the same time,the linearity,range,precision and durability of the method meet the requirements.Conclusion:The absorption and distribution of HBW-3-10 in rats were rapid,the concentration of drugs into blood was large,and the elimination was slow,and the high level of blood drug concentration could be maintained for a long time.The pharmacokinetic characteristics of HBW-3-10 in rats were preliminarily understood.After intragastric administration,HBW-3-10 can pass through the BBB and blood-cerebrospinal fluid barrier,and maintain a certain concentration in the brain tissue for a long time,which is beneficial to exert the central pharmacodynamics.This study not only enriched the content of pharmacokinetic study of HBW-3-10,but also provided a basis for new drug development and clinical scientific drug use guidance.The methodological study of HBW-3-10 provides a reference for the study of relevant substances and the establishment of quality standards for this API.