High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Microsomes Containing Recombinant Human Cytochromes P450
2.3. Pooled Human Liver Microsomes
2.4. High-Throughput Assay of P450-Dependent Demethylation
2.5. Methods of Data Analysis
2.6. S-Ketamine Demethylation Assays with LC-MS/MS Technique
3. Results and Discussion
3.1. Developing a Method for High-Throughput Assay of Cytochrome P450-Dependent Demethylation Reactions
3.1.1. Selecting the Method for Formaldehyde Detection
3.1.2. Elaborating a Method for Detecting Cytochrome-P450 Dependent Generation of Formaldehyde with the AAA Reagent
3.1.3. High-Throughput Implementation of the AAA Method
3.2. Applying AAA-Based Technique to Study Ketamine Demethylation by Human Cytochrome P450 Species
3.2.1. Setting up and Validating the AAA Method Applied to Ketamine Demethylation
3.2.2. Metabolism of Ketamine by Major Human Cytochrome P450 Species
3.2.3. N-Demethylation of S-Ketamine by Human Liver Microsomes
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Parameter | CYP3A4 | CYP2B6 | ||
---|---|---|---|---|
FA Detection | LC-MS/MS | FA Detection | LC-MS/MS | |
VMAX, min−1 | 62.8 ± 2.2 | 40.6 ± 3.6 | 50.9 ± 2.9 | 43.9 ± 2.7 |
KM, µM | 136.4 ± 13.2 | 107.3 ± 23.4 | 47.5 ± 10.6 | 36.9 ± 7.5 |
P450 Species | This Study | Yanagihara et al., 2001 [30] | Portmann et al., 2010 [31] | ||||
---|---|---|---|---|---|---|---|
KM, µM | VMAX, min−1 | CLint, µM−1min−1 | KM, µM | VMAX, min−1 | KM, µM | VMAX, min−1 | |
CYP2B6 | 53.9 ± 14.4 | 50.6 ± 8.0 | 0.938 | 44.0 ± 9.6 | 33.0 ± 3.0 | 11.9 | 26.0 |
CYP2C19 | 19.3 ± 6.6 | 13.9 ± 7.1 | 0.719 | ||||
CYP3A4 | 113 ± 18 | 44.0 ± 7.7 | 0.390 | 399 ± 48 | 42.0 ± 14.0 | 61.2 | 39.0 |
CYP3A5 | 103 ± 22 | 27.5 ± 3.7 | 0.267 | ||||
CYP2D6 | 685 ± 124 | 68.0 ± 24.3 | 0.099 | ||||
CYP2A6 | 78.6 ± 29.9 | 5.6 ± 1.6 | 0.071 | ||||
CYP1A2 | 236 ± 48 | 9.5 ± 0.6 | 0.040 | ||||
CYP2C8 | 152 ± 49 | 5.6 ± 0.4 | 0.037 | ||||
CYP2C9 | 250 ± 76 | 6.2 ± 2.0 | 0.025 | 756 ± 85 | 43.0 ± 16.0 | ||
CYP2E1 | 457 ± 182 | 5.3 ± 2.1 | 0.012 |
HLM Lot Identifier | KM1, µM | KM2. µM | Vmax (Total), min−1 | Fraction of the Low-Affinity Component, % |
---|---|---|---|---|
EGW | 225 ± 21 | 7.6 ± 0.3 | 100 | |
CDN | 159 ± 18 | 19.0 ± 0.9 | 100 | |
DNJ | 30.4 ± 14.0 | 298 ± 23 | 26.5 ± 1.3 | 86.3 ± 1.6 |
XEN263 | 31.5 ± 9.5 | 683 ± 84 | 35.8 ± 2.6 | 73.5 ± 7.0 |
FVT | 33.0 ± 18.7 | 648 ± 40 | 64.9 ± 3.0 | 92 ± 1.2 |
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Davydova, N.Y.; Hutner, D.A.; Gaither, K.A.; Singh, D.K.; Prasad, B.; Davydov, D.R. High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism. Biology 2023, 12, 1055. https://doi.org/10.3390/biology12081055
Davydova NY, Hutner DA, Gaither KA, Singh DK, Prasad B, Davydov DR. High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism. Biology. 2023; 12(8):1055. https://doi.org/10.3390/biology12081055
Chicago/Turabian StyleDavydova, Nadezhda Y., David A. Hutner, Kari A. Gaither, Dilip Kumar Singh, Bhagwat Prasad, and Dmitri R. Davydov. 2023. "High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism" Biology 12, no. 8: 1055. https://doi.org/10.3390/biology12081055