Study On Sulfonylureas Uptake And Drug-drug Interaction With Rosuvastatin Mediated By OATP1B1/OATP1B3

Background:Type 2 diabetes mellitus is a metabolic disorder that is characterized by hyperglycemia(high blood sugar) in the context of insulin resistance and relative lack of insulin. One of the major approaches to controlling hyperglycemia is antidiabetic agents among which the second generation of sulfonylureas(SUs) is the earliest applied, most diversified and most broadly-used. Since Diabetes Mellitus is often complicated by obesity, hyperlipidemia and other related diseases, SUs are often combined with statins in order to manage the complicated conditions, which can undoubtedly increase risk of drug-drug interaction(DDI). DDI can be caused by a range of factors among which the transport process of drug is one of the most crucial determinants. Therefore, investigation on the relationship between SUs and hepatic transporters OATP1B1 / OATP1 B and mechanism of potential DDI may provide guidance to rational drug use in clinic.Objectives:The transporting characteristics of five commonly used sulfonylureas(glibenclamide,gliclazide,glimepride,glipizide,gliquidone) in liver and the relationship with OATP1B1 and OATP1B3 is evaluated by constructing high expressing OATP1B1 and OATP1B3 HEK293 cells models respectively.Add inhibitors of OATP1B1 and OATP1B3 to determine changes in SUs uptake and clarify the relationship between OATP1B1 / OATP1B3 and SUs, then explore the interaction of SUs and rosuvastatin and mechanism of the interaction. Eventually theoretical basis can be provided to rational drug use.Methods:1. Method for simultaneous determination of the five SUs by LC-MS was established for the quantitative analysis of SUs in cell lysates. The cell samples were pretreated by ethyl acetate liquid-liquid extraction.2. Induce OATP1B1/OATP1B3 high expression by treating OATP1B1-HEK293 T and OATP1B3-HEK293 T cells with sodium butyrate.3. Based on the data from cytotoxicity experiments of these five SUs against HEK293 T cells, the uptake and pharmacokinetic characteristics in OATP1B1-HEK293 T and OATP1B3-HEK293 T cells were explored respectively, and kinetic parameters including Vmax and Km were calculated and compared with those of positive control rosuvastatin, and then the relationships between the SUs and transporters OATP1B1 and OATP1B3 were explored preliminarily. Compare the uptake change before and after adding OATP1B1 inhibitors BSP, rifampicin and OATP1B3 inhibitors glycyrrhizic acid, rifampicin into SUs transported by OATP1B1 and OATP1B3 and further clarify uptake mechanism of SUs’.4. Co-administrated rosuvastatin with SUs, compare the change of drug concentrations in cells.The potential mechanism of DDI between SUs and statins was further clarified.Results:1. Specificity, sensitivity, accuracy and precision of the LC-MS detection methods constructed meet test requirements well.2. The uptake of rosuvastatin, substrate of OATP1B1-HEK293 T and OATP1B3-HEK293 T,increased significantly after incubating with 1 m M sodium butyrate for 48 h, which proved that the induce model was successfully conducted for the following uptake experiment.3. Conspicuous uptake of gliclazide and glimepiride were observed in OATP1B1-HEK293 T cells, Km,Vmax were 30.18μΜ, 12.91 nmol·min-1·mg-1 protein and 10.01μΜ,61.71nmol·min-1·mg-1 protein, respectively. Km and Vmax of uptake of rosuvastatin into OATP1B1-HEK293 T cells were 34.54μΜ and 16.68 nmol. min-1.mg-1 protein.BSP and rifampicin,the inhibitor of OATP1B1 could suppress the uptake of gliclazide and glimepiride, IC50 values were 313.05μΜ,466.82μΜ and852.25μΜ,3.94 m M respectively. In addition, gliclazide and glimepiride might interact with the OATP1B1 substrate rosuvastatin to some extent: gliclazide and glimepiride would inhibit uptake of rosuvastatin when rosuvastatin was the substrate(IC50 were146.70μΜ and 76.25 respectively).Similarly, rosuvastatin played an inhibitory effect on uptake of gliclazide and glimepiride(IC50 were 222.36μΜ and 517.35μΜ).4. Conspicuous uptake of glibenclamide and glipizide were observed in OATP1B3- HEK293 T cells, the Km, Vmax were 15.36μΜ, 120.00 nmol. min-1. mg-1protein and 41.29μΜ, 41.98 nmol. min-1. mg-1 protein. Km and Vmax of rosuvastatin uptake into OATP1B3-HEK293 T cells were 35.21μΜ and 16.67 nmol. min-1. mg-1protein. Besides, The uptake of glibenclamide and glipizide were constrained when inhibitors of OATP1B3 glycyrrhizic acid and rifampicin were added, and IC50 were262.63μΜ, 53.51μΜ and 123.90μΜ, 50.08μΜ respectively. Moreover, uptake of glibenclamide and glipizide were suppressed when rosuvastatin was taken as the substrates(IC50 were 99.94μΜ and 898.90μΜ, respectively).Similarly, rosuvastatin might also inhibit their uptake(IC50 were 1.52 m M and 189.76μΜ).Conclusions:1. LC-MS method constructed in current study is simple and convenient, which can determine the concentration of five second generation of SUs simultaneously.2. Gliclazide and glimepiride are substrates of OATP1B1, the binding strength of glimepiride to OATP1B1 is stronger than rosuvastatin, while that of gliclazide is comparable with that of rosuvastatin. Furthermore, rifampicin plays an inhibitory effect on its uptake. Drug-drug interaction may occur when they are combined with statins.3. Glibenclamide and glipizide are substrates of OATP1B3, the bingding ability of glipizide to OATP1B3 is slightly stronger than that of rosuvastatin, while that of glibenclamide is much stronger. The inhibitor of OATP1B3 rifampicin exhibits moderate inhibition on uptake of these two anti-diabetic drugs, while another inhibitor glycyrrhizic acid show no significant inhibition. Glibenclamide displays an inhibitory effect on the uptake of rosuvastatin when administrated glibenclamide with statins, while the inhibition effect of glipizide is weaker.4. Gliquidone is neither the substrate of OATP1B1 nor that of OATP1B3, the hepatic uptake of gliquidone may not be mediated by them.