The Inhibition Of UGT Enzyme By Thyroxine And Triiodothyronine

Objective:Exogenous drug levothyroxine sodium was a routine medication for patients with thyroid cancer after surgery,which was widely used to hypothyroidism and TSH inhibition.Levothyroxine sodium existed in the body mainly by thyroxine(T4)and triiodothyronine(T3).UDP-glucuronosyltransferase(UGT)was the important biological metabolic enzyme to induce the inactivation and excretion of thyroid hormone.The purpose of this study was to explore the interaction between T4,T3 and UGT enzymes,which might lead to the drug-drug interaction mediated by UGT enzyme.Methods:Simulating the catalytic conditions of biological metabolic enzymes,the study established the incubation system of glycosylation reaction to UGT enzymes in vitro.11 kinds of commercially recombinational UGTs containing UGT1A1,UGT1A3,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B4,UGT2B7,UGT2B15 and UGT2B17 were used to independently exploration the biological parameters of UGTs.With thyroxine(T4)and triiodothyronine(T3)as inhibitors,and dimethyl sulfoxide as control group in the experiment,we used 4-methyl umbeliferone as non-specific substrate of UGT enzymes,observing the alteration of its metabolite 4-methylumbelliferone-?-D-glucuronide(4-MUG).The inhibition kinetic parameters and types of T4 and T3 for isoforms of UGT were determined by UPLC.The potential interaction to T4,T3 mediated by UGT enzyme in vitro was evaluated by in vitro-in vivo extrapolation(IVIVE).Finally,relying on the computer software to built the three dimensional space structure of UGT enzymes,completed molecular docking between inhibitor and protein.Further explained the molecular mechanism that T4 and T3 exhibited inhibition towards UGTs from the spatial configuration.Results:1.The inhibitory rate of T4 to UGT1A1,UGT1A3,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B4,UGT2B7,UGT2B15 and UGT2B17 wererespectively: 97.62%,77.81%,77.52%,89.41%,89.64%,53.1%,100%,73.37%,91.94%,97.55% and 87.71%.And the inhibition rates of T3 for UGT1A1,UGT1A3,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B4,UGT2B7,UGT2B15 and UGT2B17 were respectively: 75.21%,-9.21%,61.63%,6.66%,-17.78%,-1.51%,57.77%,5.75%,8.29%,-5.47% and 50.80%.2.The IC50 values of T4 for UGT1A1,UGT1A3,UGT1A7,UGT1A8,UGT1A10 and UGT2B7 were respectively: 5.37,16.43,14.86,8.47,18.94 and31.29?M.3.The Ki values of T4 for UGT1A1,UGT1A3,UGT1A7,UGT1A8,UGT1A10 and UGT2B7 were respectively: 1.5,2.4,11,9.6,4.8 and 3.0?M.4.T4 and substrate were competitive inhibiting the active sites of UGT1A1,UGT1A3,UGT1A7,UGT10 and UGT2B7,while T4 and substrate were non-competitively inhibiting the active site of UGT1A8.5.The concentration of T4 was greater than 0.15?M,then drug interaction mediated by UGT1A1 might occur.The concentration of T4 was greater than 0.24?M,then drug interaction mediated by UGT1A3 might occur.The concentration of T4 was greater than 1.1?M,then drug interaction mediated by UGT1A7 might occur.The concentration of T4 was greater than 0.96?M,then drug interaction mediated by UGT1A8 might occur.The concentration of T4 was greater than 0.48?M,then drug interaction mediated by UGT1A10 might occur.The concentration of T4 was greater than 0.3?M,then drug interaction mediated by UGT2B7 might occur.6.Molecular docking results show that the binding free energy of T4 and UGT1A1 was-6.72 kcal/mol,and binding free energy of T4 and UGT1A3 was-3.37kcal/mol,and binding free energy of T4 and UGT1A7 was-8.52 kcal/mol,binding free energy of T4 and UGT1A8 was-9.05 kcal/mol,binding free energy T4 and UGT2B7 was-5.02 kcal/mol.Conclusions:1.T4 exhibited obvious inhibition effect on UGT1A1,UGT1A3,UGT1A7,UGT1A8,UGT1A10 and UGT2B7,and competitively combined the active site of UGT1A1,UGT1A3,UGT1A7,UGT10 and UGT2B7,and non-competitively combined of the active site of UGT1A8.While T3 exhibited relatively weakinhibition effect even more activated effect.2.Reference in vitro evaluation standard,the threshold concentration of exogenous T4 resulting possible drug interaction calculated.Relative to the UGT1A1,UGT1A3,UGT1A7,UGT1A8,UGT1A10 and UGT2B7 was 0.15,0.24,1.10,0.96,0.48,and 0.30 mM,respectively.