Kratom (Mitragyna speciosa)
Inhibitory effect of mitragynine on human cytochrome P450 enzyme activities
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Context: To date, many findings reveal that most of the modern drugs have the ability to interact with herbal drugs.
Aims: This study was conducted to determine the inhibitory effects of mitragynine on cytochrome P450 2C9, 2D6 and 3A4 activities.
Methods and Material: The in vitro study was conducted using a high-throughput luminescence assay.
Statistical Analysis: Statistical analysis was conducted using one-way ANOVA and Dunnett's test with P < 0.05 vs. control. The IC values were calculated using the GraphPad Prism 5 (Version 5.01, GraphPad Software, Inc., USA)
Results: Assessment using recombinant enzymes showed that mitragynine gave the strongest inhibitory effect on CYP2D6 with an IC50 value of 0.45±0.33 mM, followed by CYP2C9 and CYP3A4 with IC50 values of 9.70±4.80 and 41.32±6.74 µM respectively. Positive inhibitors appropriate for CYP2C9, CYP2D6, and CYP3A4 which are sulfaphenazole, quinidine and ketoconazole were used respectively. Vmax values of CYP2C9, CYP2D6 and CYP3A4 were 0.0005, 0.01155 and 0.0137 µM luciferin formed/pmol/min respectively. Km values of CYP2C9, CYP2D6, and CYP3A4 were 32.65, 56.01, and 103.30 µM respectively. Mitragynine noncompetitively inhibits CYP2C9 and CYP2D6 activities with the Ki values of 61.48 and 12.86 µM respectively. On the other hand, mitragynine inhibits CYP3A4 competitively with a Ki value of 379.18 µM.
Conclusions: The findings of this study reveal that mitragynine might inhibit cytochrome P450 enzyme activities, specifically CYP2D6. Therefore, administration of mitragynine together with herbal or modern drugs which follow the same metabolic pathway may contribute to herb-drug interactions.
1 . Enzyme kinetics of CYP2C9 (id=NPDI-m-NOOw)
In Vitro Enzyme Kinetics Experiment
Metabolism was detected.
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Co-expressed
Results
Km and Vmax from Table 2
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Metabolite formation study
Commercially available
50 min
NADPH regenerating system
Not available
Available
0–100µM
2 . Enzyme kinetics of CYP2D6 (id=NPDI-o-4ZoA)
In Vitro Enzyme Kinetics Experiment
Metabolism was detected.
- CYP2D6 4173631
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Km and Vmax from Table 2
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Metabolite formation study
Commercially available
65 min
NADPH regenerating system
Not available
Available
0–100µM
3 . Enzyme kinetics of CYP3A4 (id=NPDI-M8nR1A)
In Vitro Enzyme Kinetics Experiment
Metabolism was detected.
- CYP3A4 4306811
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Co-expressed
Results
Km and Vmax from Table 2
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Metabolite formation study
Commercially available
50 min
NADPH regenerating system
Not available
Available
0–100µM
1 . Inhibition of CYP2C9 by mitragynine (id=NPDI-1tFkCA)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — High probability of interaction if their IC50 values are less than 1 µM. If IC50 values are between 1 and 10 µM, they have moderate possibility in interaction. Low drug-drug interactions if their IC50 values are greater than 10 µM.
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Co-expressed
Results
IC50 values from Table 1, Ki values from Table 3
% enzyme inhibition versus precipitant concentration:
Values estimated from Figure 2. In some cases, SEM could not be estimated from the provided figure.
0.02 μM: -5 ± 2%
0.2 μM: 16 ± 3%
2 μM: 20 ± 8%
20 μM: 54 ± 16%
200 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Mitragynine was isolated in-house. This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Commercially available
50 min
NADPH regenerating system
Not available
Available
0.02-200 µM
2 . POS. CONTROL: Inhibition of CYP2C9 by sulfaphenazole (id=NPDI-v2Z25g)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — High probability of interaction if their IC50 values are less than 1 µM. If IC50 values are between 1 and 10 µM, they have moderate possibility in interaction. Low drug-drug interactions if their IC50 values are greater than 10 µM.
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Co-expressed
Positive control
Results
Positive control
IC50 values from Table 1
% enzyme inhibition versus precipitant concentration:
Values estimated from Figure 2. In some cases, SEM could not be estimated from the provided figure.
0.02 μM: 38 ± Unknown%
0.2 μM: 56 ± 8%
2 μM: 77 ± Unknown%
20 μM: 81 ± Unknown%
200 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Positive inhibitors which are sulfaphenazole, quinidine and ketoconazole were purchased from Sigma Chemicals (St. Louis, USA). This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Positive control
Commercially available
50 min
NADPH regenerating system
Not available
Not available
0.02-200 µM
3 . Inhibition of CYP2D6 by mitragynine (id=NPDI-_PhwAA)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — High probability of interaction if their IC50 values are less than 1 µM. If IC50 values are between 1 and 10 µM, they have moderate possibility in interaction. Low drug-drug interactions if their IC50 values are greater than 10 µM.
- CYP2D6 4173631
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
IC50 values from Table 1, Ki values from Table 3
% enzyme inhibition versus precipitant concentration:
Values estimated from Figure 2. In some cases, SEM could not be estimated from the provided figure.
0.02 μM: 43 ± Unknown%
0.2 μM: 60 ± Unknown%
2 μM: 77 ± Unknown%
20 μM: 86 ± Unknown%
200 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Mitragynine was isolated in-house. This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Commercially available
65 min
NADPH regenerating system
Not available
Available
0.02-200 µM
4 . POS. CONTROL: Inhibition of CYP2D6 by quinidine (id=NPDI-d0noGQ)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — High probability of interaction if their IC50 values are less than 1 µM. If IC50 values are between 1 and 10 µM, they have moderate possibility in interaction. Low drug-drug interactions if their IC50 values are greater than 10 µM.
- CYP2D6 4173631
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Positive control
Results
Positive control
IC50 values from Table 1
% enzyme inhibition versus precipitant concentration:
Values estimated from Figure 2. In some cases, SEM could not be estimated from the provided figure.
0.02 μM: 47 ± Unknown%
0.2 μM: 79 ± 0%
2 μM: 82 ± Unknown%
20 μM: 87 ± Unknown%
200 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Positive inhibitors which are sulfaphenazole, quinidine and ketoconazole were purchased from Sigma Chemicals (St. Louis, USA). This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Positive control
Commercially available
65 min
NADPH regenerating system
Not available
Not available
0.02-200 µM
5 . POS. CONTROL: Inhibition of CYP3A4 by ketoconazole (id=NPDI-d-iKog)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — High probability of interaction if their IC50 values are less than 1 µM. If IC50 values are between 1 and 10 µM, they have moderate possibility in interaction. Low drug-drug interactions if their IC50 values are greater than 10 µM.
- CYP3A4 4306811
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Co-expressed
Positive control
Results
Positive control
IC50 values from Table 1
% enzyme inhibition versus precipitant concentration:
Values estimated from Figure 2. In some cases, SEM could not be estimated from the provided figure.
0.02 μM: 37 ± Unknown%
0.2 μM: 71 ± 3%
2 μM: 77 ± Unknown%
20 μM: 89 ± 7%
200 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Positive inhibitors which are sulfaphenazole, quinidine and ketoconazole were purchased from Sigma Chemicals (St. Louis, USA). This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Positive control
Commercially available
50 min
NADPH regenerating system
Not available
Not available
0.02-200 µM
6 . No inhibition of CYP3A4 by mitragynine (id=NPDI-ypADxQ)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
High probability of interaction if their IC50 values are less than 1 µM. If IC50 values are between 1 and 10 µM, they have moderate possibility in interaction. Low drug-drug interactions if their IC50 values are greater than 10 µM.
- CYP3A4 4306811
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Co-expressed
Results
IC50 values from Table 1, Ki values from Table 3
% enzyme inhibition versus precipitant concentration:
Values estimated from Figure 2. In some cases, SEM could not be estimated from the provided figure.
0.02 μM: -24 ± 0%
0.2 μM: -21 ± 5%
2 μM: -2 ± 1%
20 μM: 25 ± 2%
200 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Mitragynine was isolated in-house. This assay was carried out using the P450-Glo™ Screening Systems from Promega, USA.
Commercially available
50 min
NADPH regenerating system
Not available
Available
0.02-200 µM