Kratom (Mitragyna speciosa)
Inhibitory effect of mitragynine on human cytochrome P450 enzyme activities CSV JSON

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.

Kratom (Mitragyna speciosa)

24174816

2013651136

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.

mitragynine

luciferin

  • 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.

sulfaphenazole

luciferin

  • 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.

mitragynine

luciferin

  • 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.

quinidine

luciferin

  • 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.

ketoconazole

luciferin

  • 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.

mitragynine

luciferin

  • 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

1 . Enzyme kinetics of CYP2C9 (id=NPDI-m-NOOw)

In Vitro Enzyme Kinetics Experiment

Metabolism was detected.

luciferin

  • 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.

luciferin

  • 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