Kratom (Mitragyna speciosa)
The inhibitory effects of mitragynine on P-glycoprotein in vitro
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Mitragynine is a major component isolated from Mitragyna speciosa Korth or kratom, a medicinal plant known for its opiate-like and euphoric properties. Multiple toxicity and fatal cases involving mitragynine or kratom have been reported but the underlying causes remain unclear. P-glycoprotein (P-gp) is a multidrug transporter which modulates the pharmacokinetics of xenobiotics and plays a key role in mediating drug-drug interactions. This study investigated the effects of mitragynine on P-gp transport activity, mRNA, and protein expression in Caco-2 cells using molecular docking, bidirectional assay, RT-qPCR, Western blot analysis, and immunocytochemistry techniques, respectively. Molecular docking simulation revealed that mitragynine interacts with important residues at the nucleotide binding domain (NBD) site of the P-gp structure but not with the residues from the substrate binding site. This was consistent with subsequent experimental work as mitragynine exhibited low permeability across the cell monolayer but inhibited digoxin transport at 10 μM, similar to quinidine. The reduction of P-gp activity in vitro was further contributed by the downregulation of mRNA and protein expression of P-gp. In summary, mitragynine is likely a P-gp inhibitor in vitro but not a substrate. Hence, concurrent administration of mitragynine-containing kratom products with psychoactive drugs which are P-gp substrates may lead to clinically significant toxicity. Further clinical study to prove this point is needed.
1 . Induction of P-gp protein expression by 0.001 µM rifampicin (id=NPDI-GETspg)
In Vitro Transporter Induction Experiment
Induction was detected. Cutoff used —
p < 0.05 compared with control
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Protein expression
0.001 µM
72 h
Western blot
2 . Induction of P-gp protein expression by 0.01 µM rifampicin (id=NPDI-U0t5zQ)
In Vitro Transporter Induction Experiment
Induction was detected. Cutoff used —
p < 0.05 compared with control
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Protein expression
0.01 µM
72 h
Western blot
3 . Induction of P-gp protein expression by 0.1 µM rifampicin (id=NPDI-gdI_mg)
In Vitro Transporter Induction Experiment
Induction was detected. Cutoff used —
p < 0.05 compared with control
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Protein expression
0.1 µM
72 h
Western blot
4 . Induction of P-gp protein expression by 1 µM rifampicin (id=NPDI-4k7d3A)
In Vitro Transporter Induction Experiment
Induction was detected. Cutoff used —
p < 0.05 compared with control
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Protein expression
1 µM
72 h
Western blot
5 . Induction of P-gp protein expression by 10 µM rifampicin (id=NPDI-8GmUTQ)
In Vitro Transporter Induction Experiment
Induction was detected. Cutoff used —
p < 0.05 compared with control
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Protein expression
10 µM
72 h
Western blot
6 . Down regulation of P-gp mRNA expression by 1 µM mitragynine (id=NPDI-nWIYpA)
In Vitro Transporter Induction Experiment
Down Regulation was detected. Cutoff used —
p < 0.05 compared with control
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
Estimate from Fig 3a
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
mRNA expression
1 µM
72 h
Reverse transcription-quantitative real-time PCR
7 . Down regulation of P-gp mRNA expression by 10 µM mitragynine (id=NPDI-DEOq7g)
In Vitro Transporter Induction Experiment
Down Regulation was detected. Cutoff used —
p < 0.05 compared with control
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
Mean taken from text. SD estimated from Figure 3a.
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
mRNA expression
10 µM
72 h
Reverse transcription-quantitative real-time PCR
8 . Down regulation of P-gp mRNA expression by 1 µM quinidine (id=NPDI-ye4dPw)
In Vitro Transporter Induction Experiment
Down Regulation was detected. Cutoff used —
p < 0.05 compared with control
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
% Inhibition mean taken from text. SD estimated from Figure 3b.
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
mRNA expression
1 µM
72 h
Reverse transcription-quantitative real-time PCR
1 . Permeability of mitragynine across Caco-2 monolayer cells (id=NPDI-Ue8YDg)
In Vitro Transporter Kinetics Experiment
No Transport Activity was detected. Cutoff used —
(not provided)
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
Figure 2a for Papp ratio
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
100000 cells / cm^2 per insert
Transwell® polycarbonate membrane cell culture insert
21
350 - 600 Ωcm^2
72 h
0.1, 1, 10 µM