Applications Of Immobilized Artificial Membrane Chromatography In Characterizing The Interaction Of Small Molecules With Thrombin And Predicting Drug Membrane Permeability

The affinity interactions with target and themembrane permeability of drug candidates have received significant attention during drug discovery.Therefore,developing efficient methods to explore the interaction between small moleculars and receptors or enzymes is an important research area.And the study of membrane permeability or drug-membrane interactions facilitates the assessment of the passive transport of drugs and chemicals'bioaccumulation.In this thesis,a simple protein immobilization method was developed to immobilize thrombin?THR?,and then the interactions between immobilized THR and small molecule compoundswere charaterized.On the other hand,a method was proposed for the determination of the phospholipid-water sorption coefficient(log K_PLIPW),and several mathematic models were constructed to predict Caco-2 cell permeability and oral absorption based on log K_PLIPW.This thesis mainly includes five parts?charpters?:The first part is a literature review.Immobilized artificial membrane chromatography,including the properties of the IAM columns and the retention behavior of the compounds,was briefly summarized.The applications of IAM-HPLC in immobilized membrane receptor,permeability prediction and environmental risk assessment were reviewed.And at last three main methods for predicting small intestinal permeability of drugs were also introduced.In the second part,a simple method to immobilize thrombin was developed,where IAM column was used as carrier material for entrapping THR.Then the frontal analysis for five compounds was performed on the THR-IAM column.The dissociation constants were determined and the values were 2.46,12.42,44.93,76.91and 100.69?mol/L for gallic acid,protocatechuic acid,ferulic acid,chlorogenic acid and sinapic acid,respectively.In addition,molecular docking was further applied to study the interaction between the compounds and thrombin,and the ranking order of docking energy values was similar to that of the dissociation constants of compounds determined by FAC.The results of present study demonstrate that the interaction between compounds and thrombin can be well characterized by FAC experiments based on THR-IAM column along with molecular docking analysis.A mathematic model by characterizing the confounding electrostatic effects on IAM surface was developed in the third part.Based on the developed electrostatic model,much more accurate log K_PLIPW values orthe intrinsic phospholipid-water sorption coefficients(log K_IAM,intr)for diverse analytes were measured.The results showed that the confuounding electrostatic effect could be negligible for electrically neutral compounds?including some zwitterions?,while additional electrostatic repulsion/attraction interactions with the charged IAM surface were characterized for organic cations/anions.Generally,log K_PLIPWLIPW would be subtracted by 0.28 log unitson the basis of apparent phospholipid-water sorption coefficient(log K_IAM,app)for cations,with the ionic strength of 0.1 mol/L and the pH value of 7.In contrast,log K_PLIPW was equal to the K_IAM,appAM,app value plus 0.28 log units for anionsFor orally absorbed drugs,the drug permeability is a major factor during the drug absorption process.In the fourth part,the correlations between log K_IAM,app/log K_IAM,intr and intrinsic Caco-2 cell permeability(log P_Caco-2,intr)of 28 compounds were investigated.The IAM data which could be converted to log K_IAM,app/log K_IAM,intrand logP_Caco-2,intr for 28 compounds were from the literature sources.A good sigmoidal correlation?R²=0.76?was established between log K_IAM,intr and log P_Caco-2,intr for organic cations and neutral compounds.At the same time,by collecting human oral absorption data of 130 drugs from the literature,a stable mathematic model between log K_IAM,intr and oral absorption data was established.The IAM data for 130 drugs were predicted by an online log k_IAM calculator.Finally,the IAM retention data of 19drugs were determined to verify the established oral absorption model and most drugs were within the reasonable scope.The fifth part presents the summary and prospects of the present study.The results showed that IAM chromatography could be used to immobilize thrombin and evaluate its interaction with compounds,which could be able to screen potential thrombin inhibitors.In addition,the calculation method of the log K_IAM,intr proposed in this thesis was of significance for obtaining more accurate phospholipophilicity values.The mathematical coreelations of log K_IAM,intr and Caco-2 cell permeability or human oral absorption was constructed,to provide a theoretical basis for predicting the drugs permeability.Besides,IAM chromatography has good application prospectsin the protein immobilization and membrane permeability prediction.