Research On Target Occupancy Model Based On Intestinal Target Drug-target Binding Kinetics

Objective:Pancreatic lipase(EC 3.1.1.3)and α-glucosidase(E.3.2.1.20)are intestinal targeted enzymes,the former digests fat in the duodenum and the latter is located in the brush border epithelial cells of the small intestine,both are currently widely studied drug targets.Existing research shows that many compounds have good pancreatic lipase or α-glucosidase inhibitory activity(i.e.,high affinity)in in vitro screening,but the inhibitory activity in vivo is not ideal,the reason is that the limited target occupancy rate(TO)is one of the main influencing factors.The binding and dissociation rate constants of binary drug-target complexes are the key factors affecting TO,therefore,the binding kinetics(BK)attribute of compound-target interactions has gradually become a key parameter for evaluating potential pharmacodynamic activity.At the same time,when establishing a link between the target BK rate constant and TO in vivo,the concentration of free compounds in the vicinity of the target also needs to be considered.Therefore,this study will use pancreatic lipase and α-glucosidase as the research targets,use the marketed drugs that directly act on these two targets,and several traditional Chinese medicine compounds with clear inhibitory effects as the tool drugs,BK parameters are obtained through in vitro biochemical technology,the intestinal cavity pharmacokinetic(IPK)model was used to obtain the free drug concentration curve near the pancreatic lipase target,and the unstirred water layer pharmacokinetic(TPK)model was used to obtain the free drug concentration near the α-glucosidase target.Finally,combine the BK of the compound with IPK/TPK to develop a novel simulation model of target occupancy to quantify the dynamic change of target occupancy over time.Methods:Establish an in vitro enzymatic reaction system of pancreatic lipase andα-glucosidase;the in vitro enzymatic reaction system was used to determine the half inhibitory concentration(IC50)of each inhibitor of pancreatic lipase and α-glucosidase;the inhibition kinetics was investigated using Lineweaver-Burk method;the apparent rate constant kobs values were determined by measuring the reaction progress curve;the type of inhibitor binding was determined by plotting the relationship between kobs and inhibitor concentration[I];the reversibility of inhibition of a selected inhibitor was determined by measuring the recovery of enzyme activity or plotting the relationship between enzyme concentration[E]and[I];rapid dilution method or surface plasmon resonance(SPR)technique are used to determine the drug-enzyme dissociation rate constant(koff);other key combined dynamic parameters were solved by using nonlinear fitting;the IPK model and TPK model were used to calculate the drug concentration in the intestine and near the unstirred water layer at different times;to develop an in vivo target occupancy model and to calculate the target occupancy at different times in the body.Results:(1)Pancreatic lipase and α-glucosidase drug-target binding affinity studies measured the IC50 of the pancreatic lipase inhibitor orlistat to be 19.1 nM,and the IC50 of cetilistat to 76.0 nM;the IC50 of the α-glucosidase inhibitor acarbose is 0.008 × 10-3 μM,the IC50 of miglitol is 0.720×102 μM,the IC50 of deoxynojirimycin is 0.731 μM,the IC50 of EGCG is 0.405 μM,the IC50 of GCG is 0.243 μM,and the IC50 of ECG is 1.454 μM;acarbose,miglitol,deoxynojirimycin are competitive inhibitors,EGCG,GCG,and ECG are mixed inhibitors,and through exclusive studies,EGCG,GCG,and ECG may have a common binding site on the target of α-glucosidase;(2)The results of the reaction progress curve show that Orlistat,cetilistat and pancreatic lipase are time-and concentration-dependent slow binding reactions,and the binding reaction belongs to the two-step binding mode,and the binding type is mechanism B;the reaction progress curve combined with the results of the pre-incubation test shows that acarbose,ECG,EGCG and α-glucosidase are time-and concentration-dependent slow-binding reactions,and the binding reaction belongs to the two-step binding mode,the binding type is mechanism B;however,GCG,miglitol,and deoxynojirimycin are only concentration-dependent and not time-dependent,so they are not types of slow-binding reactions;(3)The reversibility study results show that orlistat and cetilistat are irreversible inhibitors of pancreatic lipase or reversible inhibitors with extremely slow dissociation rate;acarbose,miglitol,deoxynojirimycin,EGCG,GCG and ECG are reversible inhibitors ofα-glucosidase;the fast dilution method measured the binding rate constants(kon)of orlistat and cetilistat are 76.5 × 104 and 1.7 × 104 M-1S-1,and koff are 0.3 × 10-6 and 8.4 × 10-6 S-1 respectively;SPR measured the kon of acarbose,miglitol,deoxynojirimycin,EGCG,GCG and ECG are 12.91 × 104,81.13 × 10,49.90 × 103,56.67 × 102,35.80× 102 and 62.51×102 M-1S-1,and koff are 0.32 × 10-2,1.02 × 10-2,2.51 × 10-2,0.38 × 10-2,0.62 × 10-2 and 0.73 × 10-2 S-1 respectively;(4)The time when the target occupancy rate of orlistat and cetilistat and pancreatic lipase reached more than 90%were 0.08 h and 0.72 h,respectively.The predicted maximum target occupancy rates were 99.8%and 93.0%,respectively.Orlistat’s target occupancy rate for pancreatic lipase is greater than 90%at 24 h,and cetilistat’s target occupancy rate is greater than 60%at 21 h;in the four different intestinal segments of rats,the percentage of maximum α-glucosidase occupancy of ECG significantly changed from 48.9%to 95.3%,and the time span of occupancy exceeding 70%was 0 to 0.64 h;while the percentage of maximum α-glucosidase occupancy of EGCG significantly from 96.0%to 99.8%,and the time span with occupancy rate over 70%was 1.5 to 8.9 h.Conclusion and significance:This study based on the traditional evaluation of pancreatic lipase and α-glucosidase inhibitory activity based on binding affinity,by combining BK and IPK/TPK of compound components,the dynamic change of target occupancy rate over time was quantified,and a new target occupancy calculation model was developed.The research results provide a novel and feasible method for evaluating the inhibitory activity of compounds on pancreatic lipase and α-glucosidase in in vivo.