Pharmacokinetics Of A Novel KCNQ Channel Opener QO-83

Kv7/KCNQ is a class of delayed rectified voltage-dependent potassium channels,of which KCNQ2/3 channel(M channel)has become a new drug target for the treatment of diseases related to abnormal excitation of neurons such as epilepsy and neuropathic pain.Compound QO-83 is a new KCNQ ion channel opener obtained from a large number of compounds designed and synthesized by high-throughput screening and structural optimization which has independent intellectual property rights.Compared with the positive control drug retigabine(RTG),this compound shows higher biological activity from the perspective of the cellular level and the overall animal model,and has better channel subtype selectivity.As an important part of pre-clinical research on innovative drugs,this study systematically studied absorption,distribution,metabolism and excretion of compound QO-83 in rats.Part Ⅰ Study on Compound QO-83 Rat Plasma DynamicsObjective: To establish and verify a liquid chromatography-mass spectrometry method for the determination of compound QO-83 in rat plasma,and apply it to the study of plasma kinetics.Method: 1.Development of mass spectrometry and chromatographic conditions for the determination of compound QO-83 in the plasma matrix based on HPLC-MS/MS technology,and full method validation was performed in accordance with FDA guidelines.2.Intravenous injection at single dose of 1 mg/kg body weight and intragastric administration at single dose of 5,10 mg/kg body weight in rats,blood was taken from the eye at different time points,and the blood drug concentration was measured to obtain pharmacokinetic parameters and calculate the absolute bioavailability.Result: 1.Methodological verification showed that the precision,accuracy,linearity,matrix effect,extraction recovery and stability of compound QO-83 in the plasma matrix met the requirements.2.After tail vein administration at single-dose of 1 mg/kg body weight and administration at single-dose of 5,10 mg/kg body weight,the Cmax of the drug in the rats is 1196.3,49.217,and 147.67 ng/m L,respectively.AUC(0-t)is 1099.9,217.02,680.13 ug/L*h,respectively.AUC(0-∞)is 1115.8,222.24,727.46 ug/L*h,respectively.t1/2 is 2.478 h,and absolute bioavailability is 5.06 ± 1.58%.Conclusion: The LC-MS method for determining the content of compound QO-83 in the plasma matrix of rats established in this experiment is fast,accurate,and highly sensitive,and can meet the detection requirements.Compound QO-83 has poor oral absorption,low bioavailability,moderate half-life,and new formulations need to be developed.Part Ⅱ Study on Tissue Distribution of Compound QO-83 in RatsObjective: To establish and verify the LC-MS method for determining the content of compound QO-83 in the homogenate matrix of rat tissues,and to apply it to the study of the distribution of compound QO-83.Method: 1.Developed mass spectrometry and chromatographic conditions for the determination of compound QO-83 in each tissue homogenate matrix based on HPLC-MS/MS technology,and carried out full methodological verification in liver tissue homogenates according to FDA guidelines;Partial verification of key items was performed in other tissue homogenates.2.After intragastric administration at a single dose of 10mg/kg body weight,all tested animals were anesthetized killed and collected heart,liver,spleen,lung,kidney,brain,stomach,fat,bladder tissues and blood at 15 min,1 h,8 h,respectively.Determine the drug content of compound QO-83 in each tissue and plasma,investigate the distribution level of the drug in various tissues of rats.Result: 1.Taking liver tissue as an example,methodological verification shows that the precision,accuracy,linearity,matrix effect,extraction recovery and stability of compound QO-83 meet the requirements,and the key verification items of other tissues met the requirements.2.After intragastric administration for 15 minutes,higher levels of drug concentration can be detected in various tissue organs,with a concentration range of 54.93 ~ 1230.83 ng/g;At 1 h,the drug concentration of all tissues increased(except for gastric tissue),and the concentration range was 74.38 ~ 747.33 ng/g;After 8 h,the drug concentration of all tissues and organs was low,ranging from 2.64 to 534.75 ng/g(except for gastric tissue).The levels of drugs in brain tissue were higher than the plasma levels at the same time,indicating that the compound QO-83 easily passed the blood-brain barrier.Conclusion: The LC-MS method for determining the content of compound QO-83 in the biological matrix of rat tissues established in this experiment is fast,accurate,and highly sensitive,and can meet the detection requirements.The compound QO-83 is rapidly and widely distributed in animals,and easily penetrates the blood-brain barrier,with no obvious tissue accumulation.Part Ⅲ Study on Excretion Kinetics of Compound QO-83 in RatsObjective: To establish and verify the LC-MS method for determining the content of compound QO-83 in feces,urine and bile matrix of rats,and to apply it to the study of excretion of compound QO-83.Method: 1.Development of mass spectrometry and chromatographic conditions for the determination of compound QO-83 in feces,urine and bile matrix based on HPLC-MS/MS technology,and full methodological verification according to FDA guidelines.2.Intragastric administration at a single dose of 10 mg/kg body weight,measured the content of the original drug in feces,bile and urine and investigated the excretion pathways and processes of prototype drug in rats.Result: 1.Methodological verification showed that the precision,accuracy,linearity,matrix effect,extraction recovery and stability of the compound QO-83 in feces,urine and bile met the requirements.2.The cumulative excretion of compound QO-83 in feces and bile is 1.14 mg,which is equivalent to 56.63% of the dose,suggesting that the compound QO-83 have been metabolized or excreted by other routes.The cumulative excretion rate of the drug in feces was 56.563%.The cumulative excretion rate of the drug in bile was 0.067%.No prototype drug was detected in urine.Conclusion: The LC-MS method for determining the content of compound QO-83 in feces,urine and bile of rats established in this experiment is fast,accurate,and highly sensitive,and can meet the detection requirements.Half of the drug is excreted from feces and bile in the prototype form,and the remaining half is presumed to be excreted in the form of metabolites.Part Ⅳ Metabolism of Compound QO-83 in RatsObjective: To analyze and identify the metabolites of compound QO-83 in bile,plasma,urine and feces in rats based on LC/MS.Method: 1.Bile,plasma,urine and feces samples were concentrated and processed by liquid-liquid extraction.2.HPLC-QTOF-MS method and HPLC-QTrap-MS combined with Lightsight software method were established to analyze the metabolites in bile,plasma,urine and feces after administration.3.After data collection is complete,Metabolitepilot 1.5,Peakview 2.2,Chemdraw 14.0 and other professional software were used in combination with drug metabolism and chemical cleavage rules for analysis and identification.Result: Based on the law of metabolic transformation of the drug in the body and the cleavage law summarized by the parent drug,6 phase I and 17 phase II metabolites were presumed.The main types of metabolic reactions include hydroxylation,ketolation,glucuronidation,sulfonation,methylation, acetylation,phosphorylation,amino acid binding(glycine,glutamine,cysteine,taurine).conclusion: The metabolite identification technology strategy based on the combination of HPLC-QTOF-MS and HPLC-QTrap-MS has good specificity,high sensitivity,high efficiency and reliable results,which can be used for the preliminary identification of metabolites conveniently and quickly.A total of 23 metabolites of the compound QO-83 were detected in this experiment,which preliminary revealed the metabolic rules.