| Background:In our preliminary studies, it was observed that UA was metabolized less than30% in nicotinamide adenine dinucleotide phosphate(NADPH)-HLM incubation system and was metabolized about 80% in UGT assay. It indicated that phase Ⅱmetabolism mediated by UGTs may be the major metabolic pathways. The purpose of this study is to characterize the glucuronidation kinetics of UA in HLMs and identify the main UGTs isoforms involved using a battery of recombinant human UGTs and provide experimental basis or theoretical support in the metabolic behavior of UA in humans for further study.Objectives:The purpose of this study is to characterize the glucuronidation kinetics of UA in HLMs or HIMs and identify the main UGTs isoforms involved using a battery of recombinant human UGTs. Inhibition of UGT-glucuronidation activity and correlation in HLMs were also determined using known high-affinity UGT substrates or inhibitors to facilitate identification of the UGT isoforms involved in UA glucuronidation.Methods:1. To study the glucuronidation of UA mediated by UGTs in HLMs incubation system.1 To establish the optimal incubase protein concentration of pooled HLMs and the optimum incubase time of UA in UDPGA- HLMs system, finally to establish in the incubation method of UA and the sample processing methods in UDPGA-HLMs system.2 To establish a sensitive and feasible liquid chromatography-tandem mass spectrometry analytical method for the determination of UA and the metabolites.3 To research the correlation between the concentration of UA and the incubation time, and the correlation between the metabolites of UA and the concentration of UA in HLMs.4 To study the hydrolysis of the glucuronidation of UA with β-glucuronidase in HLMs.5 A serious concentration of UA were incubated in UDPGA-HLMs system,and measuring the concentration of UA, and fitting the enzyme kinetics curve, finally calculating the enzyme kinetics parameters of Km, Vmax, Clint.2. To research the glucuronidation of UA mediated by UGTs in HIMs incubation system.A serious concentration of UA were incubated in UDPGA- HIMs system,and measuring the concentration of UA, and fitting the enzyme kinetics curve, finally calculating the enzyme kinetics parameters of Km, Vmax, Clint.3. To study the glucuronidation of UA in recombinant UGTs.1 Twelve commercially available combination human UGTs were used to screen the glucuronidation of UA at three concentrations(4, 8, and 16 μM).Incubation conditions and analyses were similar to those HLMs except that the protein concentration was 0.2 mg/ml.2 A serious concentration of UA were incubated in combination human UGTs system,and measuring the concentration of UA, and fitting the enzyme kinetics curve,finally calculating the enzyme kinetics parameters of Km, Vmax, Clint.4. Inhibition analysis of UA glucuronidation in HLMsThe inhibitory effects of known UGT isoform-selective inhibitors on the formation of UA glucuronide were evaluated to identify the UGT isoforms involved in the metabolic pathway. Inhibitors used in the current study were as follows:chenodeoxycholic acid(0,10,20,50 μM) for UGT1A3, hecogenin(0,10,20,50 μM) for UGT1A4. The formation rates of UA glucuronide from UA(4 μM) were determined from reaction mixtures incubated in the presence or absence of inhibitors. With the exception of adding UGT-isoform-specific inhibitors, all other incubation conditions were described as above. The glucuronidation activities were calculated as a percentage of control, and the inhibitory concentration(IC50) indicating that the concentration inhibits 50% of the control concentration, and were calculated by nonlinear curve fitting with Graphpad Prism.5. Correlation analysis by individual HLMsLinear regression analysis was used to determine the correlation between the glucuronidation of UA and either telmisartan or TFP by eight individual HLM.UGT1A3 catalyzed telmisartan and UGT1A4 catalyzed TFP glucuronidation were assessed by determining the rates of metabolite formation as previously reported.Reaction mixtures were incubated for 30 min at 37°C using 0.5 mg/ml of HLM protein and UA, telmisartan, TFP concentrations of 4, 2, and 10 μM, respectively. The correlation coefficient(r) was calculated to estimate the relationship between glucuronidation activities of UA in individual HLMs and telmisartan for UGT1A3 or TFP for UGT1A4.Results:1. Identify the glucuronidation of UA mediated by UGTs in HLMs incubation system1 The UA incubate system were incubated with different concentrations(0.25,0.5,1.0,1.5mg/ml) of HLMs for 30 min, it showed the metabolic rates of UA in the 0.5mg/ml HLMs incubation system was about 30%. The UA incubate system contained 0.5mg/ml HLMs was incubated for 15,30,45,60,90 min after the reaction was initiated at optimal incubate time of UA incubate system. It showed the metabolic rate of UA was about 30%. So the optimal incubate protein concentration of HLMs and the optimal incubate time of the UA incubation reaction was 0.5mg/ml is0.5mg/m and 30 min.2 The mass spectrum of metabolite was dominated by [M-H]-ion at m/z631.30, corresponding to UA glucuronide, and followed by an ion at m/z 455.30,corresponding to the parent drug UA-H with characteristic m/z 176 loss of the GA moiety. The MS/MS spectra contain the characteristic fragment ions of UA at m/z(174.9 and 112.9), which demonstrates the presence of a GA moiety. The ion at m/z112.9 was formed by further dissociation of the ion at m/z 174.9 and was the common feature ion in the negative ion mass spectra of glucuronide metabolites.3 We can find the result from curves of time and concentration that the relationship between the decrease of UA concentration and the increase incubation time, between the increase of glucuronic acid products of UA and the increase of UA substrate concentration is according with positive correlation.4 UA glucuronide can be hydrolyzed by β-glucuronidase and converted into parent UA.In the sample of hydrolysates by β-D-glucuronidase we can find that glucuronide of UA has disappeared, the content of UA has increased with glucuronide of UA to reduce consistent.5 Kinetic analysis of UA glucuronidation was investigated in HLMs. The substrate concentration of glucuronidation velocity curves showed typical Michaelis-Menten kinetics.Incubation of UA from 1 to 64 μM with HLMs revealed that the Km, Vmax, and Clint(intrinsic clearance) values(Vmax/Km) for UA glucuronide were 3.29±0.16 μM, 0.33±0.03 nmol/min(/mg protein), and 0.10 μl/min/(mg protein),respectively.2. Kinetic analysis of UA glucuronidation was investigated in HIMs. The substrate concentration of glucuronidation velocity curves showed typical MichaelisMenten kinetics.Incubation of UA from 0.25 to 16 μM with HLMs revealed that the Km, Vmax, and Clint(intrinsic clearance) values(Vmax/Km) for UA glucuronide were5.38±0.45 μM, 0.39±0.04 nmol/min/(mg protein),0.07 μl/min/(mg protein),respectively.3. To study the glucuronidation of UA in recombinant UGTs.1 Twelve recombinant UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8,1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 were applied to catalyze UA glucuronidation and to identify the isozymes involved in the metabolism. All three UA concentrations(4, 8, and 16 μM), UGT1A3, and UGT1A4 were displayed the highest and most prominent catalytic activity to UA glucuronidation.2 The kinetic parameters fitting the data points to the Michaelis-Menten equation was displayed(Table 1). Incubation of UA(1 to 64 μM) with recombinant UGT1A3 and UGT1A4 revealed that the Km, Vmax, and Clint values for UA glucuronide were 2.58±0.12 and 4.66±0.60 μM, 0.72±0.01 and 1.00±0.06nmol/min/mg proteins, and 0.28 and 0.21 μl/min/mg proteins, respectively. This result demonstrated that the UGT1A3 and UGT1A4 are the most important isoforms involved in the UA glucuronidation.4. Inhibition analysis of UA glucuronidation in HLMs.The inhibitory effects of chenodeoxycholic acid(UGT1A3) and hecogenin(UGT1A4) on UA glucuronidation were investigated in HLMs. Glucuronidation activities in HLMs were reduced by inhibitors with a concentration-dependent manner.When chenodeoxycholic acid and hecogenin were added together, it exhibited strong inhibition of UA in HLMs. The UA UGT activity was inhibited by chenodeoxycholic acid(IC50=28.26±2.9 μM) and hecogenin(IC50=51.79±4.3 μM)5. To further assess the contribution of UGT isoforms, correlation analyses were performed between the UA UGT activity and UGT1A3 or UGT1A4 probe substrate telmisartan and TFP by individual HLMs. There was a significant correlation between UA and telmisartan glucuronidation(r=0.8752, p<0.01) and a medium correlation between UA and TFP glucuronidation(r=0.7659, p<0.01).Conclusions:UA was metabolized by glucuronidation in UDPGA-HLMs and UDPGA-HIMs incubation system, and UGT1A3 and UGT1A4 enzyme were the main metabolic enzymes responsible for UA glucuronidation reactions. Chenodeoxycholic acid(UGT1A3 inhibitor) and hecogenin(specific inhibitor of UGT1A4) all have an inhibitory effect for glucuronidation reaction in UDPGA-HLMs incubatin system.There was a more significant correlation between UGT1A3 probe substrates telmisartan and glucuronidation metabolism of UA, and there was a medium correction between the UGT1A4 probe substrates TFP and glucuronidation metabolism of UA. These results further suggested that UGT1A3 and UGT1A4 were the main isoform enzymes responsible for the glucuronidation of UA in human. |
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