Kratom (Mitragyna speciosa)
In vitro evaluation of cytochrome P450 induction and the inhibition potential of mitragynine, a stimulant alkaloid CSV JSON

CYP450 enzymes are key determinants in drug toxicities, reduced pharmacological effect and adverse drug reactions. Mitragynine, an euphoric compound was evaluated for its effects on the expression of mRNAs encoding CYP1A2, CYP2D6 and CYP3A4 and protein expression and resultant enzymatic activity. The mRNA and protein expression of CYP450 isoforms were carried out using an optimized multiplex qRT-PCR assay and Western blot analysis. CYP1A2 and CYP3A4 enzyme activities were evaluated using P450-GloTM assays. The effects of mitragynine on human CYP3A4 protein expression were determined using an optimized hCYP3A4–HepG2 cell-based assay. An in silico computational method to predict the binding conformation of mitragynine to the active site of the CYP3A4 enzyme was performed and further validated using in vitro CYP3A4 inhibition assays. Mitragynine was found to induce mRNA and protein expression of CYP1A2. For the highest concentration of 25 lM, induction of mRNA was approximately 70% that of the positive control and was consistent with the increased CYP1A2 enzymatic activity. Thus, mitragynine is a significant in vitro CYP1A2 inducer. However, it appeared to be a weak CYP3A4 inducer at the transcriptional level and a weak CYP3A4 enzyme inhibitor. It is therefore, unlikely to have any signif- icant clinical effects on CYP3A4 activity.

Kratom (Mitragyna speciosa)

PMID:23274770

23274770

1 . Unnamed Experiment (id=NPDI-7sdBtQ)

In Vitro Enzyme Induction Experiment

Induction was detected.  Cutoff used — Not specified

mitragynine

  • CYP1A2 4312402

Cell system HepG2 cell line

Figure 2

Results

68.5% of induction compared to positive control at [25 uM]

Sample Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

mRNA expression

0.1, 0.5, 1, 5, 10, 25 uM

48 hours

Control Experiment Information

2 . Unnamed Experiment (id=NPDI-Ww9TTQ)

In Vitro Enzyme Induction Experiment

Induction was detected.  Cutoff used — Not specified

mitragynine

  • CYP2D6 4173631

Cell system HepG2 cell line

Figure 3

Results

CYP2D6 protein expression was found to be increased within the range of 1–5 uM of mitragynine relative to the untreated control. [...] Although the corresponding mRNA expression of CYP2D6 did show slight increase in the mRNA levels, the experiments were not statistically significant for all treatment conditions

Sample Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

Protein expression

0.1, 0.5, 1, 5, 10, 25 uM

48 hours

Control Experiment Information

3 . Unnamed Experiment (id=NPDI-LD3MZA)

In Vitro Enzyme Induction Experiment

Induction was detected.  Cutoff used — Not specified

mitragynine

  • CYP1A2 4312402

Cell system HepG2 cell line

Figure 3

Results

the protein expression of CYP1A2 showed an increased in relative values from 0.5uM onwards as compared to control. Concentration-dependent effects was only observed from 0.1 to 1.0 uM. The mean relative intensity at 1 and 10 lM showed an increase in value, which corresponded with the positive control (omepra- zole). The effect of mitragynine on the CYP1A2 protein expression was consistent with the effect observed on CYP1A2 mRNA expression, although the concentration-dependent effects were not as obvious.

Sample Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

Protein expression

0.1, 0.5, 1, 5, 10, 25 uM

48 hours

Control Experiment Information

4 . Unnamed Experiment (id=NPDI-LcX0tA)

In Vitro Enzyme Induction Experiment

Induction was detected.  Cutoff used — Not specified

mitragynine

  • CYP3A4 4306811

Cell system HepG2 cell line

Results

"Mitragynine appeared to moderately induce the enzymatic activity of CYP3A4. The increase was gradual and the changes in the enzymatic activity were minimal. The greatest increase in CYP3A4 enzymatic activity, approximately 43.0% relative to the positive control, was observed when cells were treated with mitragynine at 25 lM (Fig. 4b)."

Sample Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

Enzyme activity

Control Experiment Information

5 . Unnamed Experiment (id=NPDI-J0Eryw)

In Vitro Enzyme Induction Experiment

Induction was detected.  Cutoff used — Not specified

mitragynine

  • CYP1A2 4312402

Cell system HepG2 cell line

Figure 4

Results

at [25 uM]

Sample Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

Enzyme activity

0.1, 0.5, 1, 5, 10, 25 uM

48 hours

Control Experiment Information

6 . Unnamed Experiment (id=NPDI-sYa6vQ)

In Vitro Enzyme Induction Experiment

Induction was detected.  Cutoff used — Not specified

mitragynine

  • CYP3A4 4306811

Cell system HepG2 cell line

Figure 3

Results

The protein expression of CYP3A4 was slightly induced after treatment with mitragynine, with higher relative protein expression at concentrations of 0.1uM and above, with the highest induction occurring at 0.5 uM relative to untreated controls

Sample Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

Protein expression

0.1, 0.5, 1, 5, 10, 25 uM

48 hours

Control Experiment Information

7 . Unnamed Experiment (id=NPDI-5_pf-w)

In Vitro Enzyme Induction Experiment

Induction was detected.  Cutoff used — Not specified

mitragynine

  • CYP3A4 4306811

Cell system HepG2 cell line

(Figure 2)
The expression of CYP3A4 was only slightly induced by mitragynine at all concentrations tested. Although this induction was statistically significant within the range of 1–5 uM of mitragynine, the induction relative to dexamethasone was generally less than 20% (18.1% and 17.5%, respectively)

Results

Sample Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

mRNA expression

0.1, 0.5, 1, 5, 10, 25 uM

48 hours

Control Experiment Information

8 . Unnamed Experiment (id=NPDI-_MysYg)

In Vitro Enzyme Induction Experiment

Non-induction was detected.  Cutoff used — Not specified

mitragynine

  • CYP2D6 4173631

Cell system HepG2 cell line

Figure 2

Results

"There was no significant change in the mRNA expression of CYP2D6 when the HepG2 cells were treated with mitragynine"

Sample Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

mRNA expression

0.1, 0.5, 1, 5, 10, 25 uM

48 hours

Control Experiment Information