Atypical Kinetics and Albumin Effect of Glucuronidation of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5- yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6- dichlorophenyl)-3H-1,2,4-triazol-3-one, a Novel Nonpeptide Angiotensin Type 1 Receptor Antagonist, in Liver Microsomes and UDP-Glucuronosyl-transferase
Abstract
:1. Introduction
2. Results
2.1. Metabolites of Ib in Microsomes
2.2. Screening by Recombinant UGTs
2.3. Binding of Ib to BSA
2.4. Ib Glucuronidation by Microsomes and BSA Effect
2.5. Ib Glucuronidation by Recombinant UGTs and BSA Effect
3. Discussion
4. Materials and Methods
4.1. Chemicals and Materials
4.2. LC-MS/MS Assay
4.3. Enzyme Incubation
4.4. Glucuronidation in Microsomes
4.5. Glucuronidation by Recombinant UGTs
4.6. Protein Binding Assay of Ib
4.7. Enzyme Kinetic Data Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
Ib | 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloro- phenyl)-3H-1,2,4-triazol-3-one |
AT1 | angiotensin type 1 |
BSA | bovine serum albumin |
IS | internal standard |
UPDGA | uridine 5’-diphosphoglucuronic acid |
NADP | β-nicotinamide adenine dinucleotide phosphate |
G-6-P | d-glucose 6-phosphate |
PDH | glucose-6-phosphate dehydrogenase |
HLM | human liver microsomes |
MKLM | monkey liver microsomes |
DLM | dog liver microsomes |
RLM | rat liver microsomes |
MLM | mouse liver microsomes |
UGT | UDP-glucuronosyltransferases |
CYP | cytochrome P450 |
LC-MS/MS | liquid chromatography-tandem mass spectrometry |
DDI | drug-drug interactions |
IVIVE | in vitro-in vivo extrapolation |
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Sample Availability: Samples of the compounds of Ib and Ib monoglucuronide are available from the authors. |
Ib, μM | 0.2% BSA | 0.5% BSA | 1% BSA | 2% BSA |
---|---|---|---|---|
10 | 0.063 ± 0.005 | 0.041 ± 0.003 | 0.022 ± 0.002 | 0.016 ± 0.001 |
20 | 0.114 ± 0.010 | 0.043 ± 0.004 | 0.024 ± 0.002 | 0.020 ± 0.001 |
50 | 0.169 ± 0.013 | 0.063 ± 0.005 | 0.026 ± 0.002 | 0.021 ± 0.002 |
100 | 0.339 ± 0.033 | 0.084 ± 0.009 | 0.053 ± 0.004 | 0.031 ± 0.002 |
200 | 0.428 ± 0.047 | 0.105 ± 0.010 | 0.095 ± 0.007 | 0.035 ± 0.003 |
400 | 0.559 ± 0.038 | 0.170 ± 0.017 | 0.112 ± 0.009 | 0.043 ± 0.005 |
600 | 0.574 ± 0.051 | 0.358 ± 0.028 | 0.117 ± 0.010 | 0.066 ± 0.007 |
800 | 0.577 ± 0.040 | 0.434 ± 0.034 | 0.150 ± 0.012 | 0.153 ± 0.008 |
2000 | 0.613 ± 0.079 | 0.495 ± 0.039 | 0.192 ± 0.015 | 0.183 ± 0.010 |
Enzyme Source | Parameter | No BSA | 0.2% BSA | 0.5% BSA | 1% BSA | 2% BSA |
---|---|---|---|---|---|---|
HLM | Km (μM) | 59.8 ± 4.6 | 5.55 ± 0.95 | 5.80 ± 1.80 | 6.10 ± 1.10 | 5.99 ± 0.93 |
Vmax (pmol/min/mg) | 179.8 ± 6.7 | 316.3 ± 33.6 | 356.8 ± 37.2 | 389.5 ± 85.3 | 357.3 ± 34.4 | |
Ki (μM) | 455.5 ± 59.9 | 300.3 ± 86.5 | 350.0 ± 106.6 | 200.3 ± 92.4 | 354.1 ± 106.5 | |
CLint (μL/min/mg) | 3.02 ± 0.31 | 57.8 ± 9.6 | 64.4 ± 14.3 | 66.0 ± 20.6 | 61.2 ± 15.4 | |
DLM | Km (μM) | 9.00 ± 2.10 | 1.48 ± 0.33 | 1.44 ± 0.20 | 1.40 ± 0.19 | 1.11 ± 0.23 |
Vmax (pmol/min/mg) | 890.0 ± 44.0 | 1712.0 ± 308.1 | 1533.0 ± 122.81 | 1492.0 ± 71.4 | 1182.0 ± 65.4 | |
Ki (μM) | 233.6 ± 54.0 | 199.0 ± 59.5 | 89.7 ± 19.9 | 74.1 ± 10.0 | 226.4 ± 20.0 | |
CLint (μL/min/mg) | 101.9 ± 19.4 | 1164.5 ± 52.8 | 1086.6 ± 38.5 | 1083.7 ± 99.9 | 1088.2 ± 70.2 | |
RLM | Km (μM) | 13.0 ± 3.1 | 1.83 ± 0.78 | 2.00 ± 0.12 | 1.92 ± 0.51 | 1.80 ± 0.40 |
Vmax (pmol/min/mg) | 28.5 ± 2.4 | 41.5 ± 2.3 | 57.2 ± 10.3 | 37.5 ± 2.0 | 36.5 ± 3.7 | |
Ki (μM) | 332.9 ± 50.3 | 187.5 ± 34.3 | 176.2 ± 84.6 | 270.0 ± 53.9 | 263.7 ± 58.0 | |
CLint (μL/min/mg) | 2.25 ± 0.36 | 25.6 ± 5.6 | 28.8 ± 6. 9 | 20.7 ± 6.8 | 20.7 ± 2.6 |
Enzyme Source | Parameter | No BSA | 0.2% BSA | 0.5% BSA | 1% BSA | 2% BSA |
---|---|---|---|---|---|---|
UGT1A9 | Km (μM) | 17.7 ± 4.6 | 20.8 ± 4.3 | 1.61 ± 0.52 | 1.23 ± 0.42 | 1.06 ± 0.21 |
Vmax (pmol/min/mg) | 1.79 ± 0.43 | 10.1 ± 4.5 | 6.39 ± 0.93 | 5.73 ± 0.70 | 3.85 ± 0.22 | |
Ki (μM) | 508.8 ± 134.4 | 236.1 ± 46.1 | 251.7 ± 38.7 | 96.6 ± 36.4 | 185.2 ± 52.8 | |
CLint (μL/min/mg) | 0.10 ± 0.03 | 0.49 ± 0.19 | 4.14 ± 0.80 | 5.52 ± 1.48 | 3.75 ± 0.92 | |
UGT2B4 | Km (μM) | 79.3 ± 9.5 | 7.40 ± 2.40 | 1.65 ± 0.28 | 3.70 ± 0.96 | 3.65 ± 0.70 |
Vmax (pmol/min/mg) | 13.6 ± 2.0 | 33.2 ± 3.4 | 43.8 ± 1.9 | 28.7 ± 2.2 | 21.5 ± 1.3 | |
Ki (μM) | 682.7 ± 148.7 | 844.0 ± 101.5 | 352.6 ± 60.6 | 321.0 ± 106.3 | 1104.0 ± 236.5 | |
CLint (μL/min/mg) | 0.17 ± 0.04 | 4.95 ± 1.18 | 27.1 ± 5.1 | 8.20 ± 2.71 | 6.09 ± 1.55 | |
UGT2B7 | Km (μM) | 21.6 ± 2.3 | 6.00 ± 1.31 | 2.51 ± 0.65 | 0.99 ± 0.30 | 0.56 ± 0.11 |
Vmax (pmol/min/mg) | 4.07 ± 0.50 | 9.63 ± 1.81 | 7.10 ± 1.04 | 5.95 ± 0.72 | 4.31 ± 0.20 | |
Ki (μM) | 478.4 ± 169.3 | 304.0 ± 153.9 | 415.6 ± 83.6 | 172.0 ± 56.5 | 146.6 ± 31.4 | |
CLint (μL/min/mg) | 0.20 ± 0.04 | 1.52 ± 0.18 | 2.66 ± 0.68 | 6.25 ± 1.22 | 8.92 ± 0.78 | |
UGT1A3 | Km (μM) | 37.6 ± 10.6 | 4.35 ± 1.37 | 1.40 ± 0.21 | 0.70 ± 0.16 | 0.75 ± 0.10 |
Vmax (pmol/min/mg) | 146.8 ± 21.2 | 124.3 ± 13.5 | 107.8 ± 6.5 | 100.1 ± 14.5 | 87.4 ± 2.7 | |
Ki (μM) | 862.7 ± 265.8 | 607.4 ± 44.6 | 499.6 ± 85.9 | 452.7 ± 154.3 | 816.5 ± 189.0 | |
CLint (μL/min/mg) | 4.19 ± 1.21 | 30.2 ± 7.8 | 77.9 ± 9.6 | 144.9 ± 22.7 | 118.3 ± 19.5 | |
UGT1A8 | Km (μM) | 25.6 ± 4.3 | 16.0 ± 5.7 | 4.73 ± 1.05 | 2.13 ± 0.34 | 1.29 ± 0.29 |
Vmax (pmol/min/mg) | 24.3 ± 3.5 | 21.9 ± 1.2 | 19.3 ± 4.9 | 18.4 ± 3.6 | 17.8 ± 1.1 | |
Ki (μM) | 606.4 ± 156.8 | 357.5 ± 91.1 | 209.6 ± 15.2 | 218.4 ± 83.7 | 179.9 ± 43.1 | |
CLint (μL/min/mg) | 0.96 ± 0.14 | 1.52 ± 0.65 | 4.16 ± 1.10 | 8.68 ± 1.48 | 14.21 ± 2.74 |
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Peng, Y.; Zhang, X.; Zhu, Y.; Wu, H.; Gu, S.; Chang, Q.; Zhou, Y.; Wang, G.; Sun, J. Atypical Kinetics and Albumin Effect of Glucuronidation of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5- yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6- dichlorophenyl)-3H-1,2,4-triazol-3-one, a Novel Nonpeptide Angiotensin Type 1 Receptor Antagonist, in Liver Microsomes and UDP-Glucuronosyl-transferase. Molecules 2018, 23, 688. https://doi.org/10.3390/molecules23030688
Peng Y, Zhang X, Zhu Y, Wu H, Gu S, Chang Q, Zhou Y, Wang G, Sun J. Atypical Kinetics and Albumin Effect of Glucuronidation of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5- yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6- dichlorophenyl)-3H-1,2,4-triazol-3-one, a Novel Nonpeptide Angiotensin Type 1 Receptor Antagonist, in Liver Microsomes and UDP-Glucuronosyl-transferase. Molecules. 2018; 23(3):688. https://doi.org/10.3390/molecules23030688
Chicago/Turabian StylePeng, Ying, Xueyuan Zhang, Yinci Zhu, Hui Wu, Shiyin Gu, Qingqing Chang, Yi Zhou, Guangji Wang, and Jianguo Sun. 2018. "Atypical Kinetics and Albumin Effect of Glucuronidation of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5- yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6- dichlorophenyl)-3H-1,2,4-triazol-3-one, a Novel Nonpeptide Angiotensin Type 1 Receptor Antagonist, in Liver Microsomes and UDP-Glucuronosyl-transferase" Molecules 23, no. 3: 688. https://doi.org/10.3390/molecules23030688